Dehydrating agent and method for dehydrating moist article using the agent and dehydrated article obtained by the method

ABSTRACT

The present invention has the object to dehydrate hydrous matters without denaturing or deteriorating them by using a dehydrating agent comprising an anhydrous cyclotetrasaccharide, and provides a dehydrating agent comprising the cyclotetrasaccharide; a method for dehydrating hydrous matters through a step of incorporating, contacting or coexisting the cyclotetrasaccharide into, with, or in the hydrous matters; and dehydrated products obtainable thereby.

TECHNICAL FIELD

The present invention relates to a dehydrating agent comprising, as aneffective ingredient, a saccharide having the structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1}(referred to as “cyclotetrasaccharide” based on the structure,throughout the specification hereinafter); a method for dehydratinghydrous matters using the same; and dehydrated products prepared by themethod.

BACKGROUND ART

As disclosed in Japanese Patent Kokai Nos. 136,240/87, 152,536/87,152,537/87, 170,221/94, etc., by the present inventors, methods fordehydrating hydrous matters using saccharides are those which exertdehydrating powers of anhydrous saccharides by allowing them to entrapmoisture and to be converted into their hydrous crystalline forms.Unlike heat drying, these methods do not require severe conditions andhave features that they convert hydrous matters into dehydrated productswithout denaturing or deteriorating them.

However, it was revealed that, among the above methods, the onedisclosed in Japanese Patent Kokai No. 152,536/87, where anhydrousaldohexoses such as anhydrous glucose and anhydrous galactose are used,has poor preservation stability of dehydrated products because, in spiteof their relatively high dehydration powers, the saccharides are highlyreactive or easily react with amino acids, peptides, etc., and causebrowning reaction. It was also found that such anhydrous aldohexoses arenot converted into any hydrous form even under a relatively high humidcondition and have only a poor dehydrating power. As for the methodsusing anhydrous maltose and palatinose, which are respectively disclosedin Japanese Patent Kokai Nos. 136,240/87 and 152,537/87, it was foundthat there still remains anxiety about stability of dehydrated productswhen preserved for a relatively long period of time, because of theirinherent reducibilities, though they are relatively low. In addition,these methods have the demerit that they require a relatively largeamount of anhydrous maltose or palatinose as a dehydrating agent becausethese saccharides have only a relatively-low-moisture-entrapping poweras low as about 5% (w/w) to each of their weights.

Since the non-reducing anhydrous glycosyl fructosides such as anhydrousraffinose, anhydrous erlose, and anhydrous melezitose, which aredisclosed in Japanese Patent Kokai No. 152,537/87, have no reducingpower, these saccharides would be neither react with amino acids andpeptides nor cause browning reaction, and they also have advantageousstability for a relatively long period of time. The above saccharides,however, have an intramolecular fructoside bond poor in acid tolerance,speculating that they should not necessarily be appropriately used asdehydrating agents for acid hydrous matters. Accordingly, there stillremains anxiety about the stability of dehydrated products producedthereby. While anhydrous α,α-trehalose, disclosed in Japanese PatentKokai No. 170,221/94, has no reducing power and satisfactory stabilizesdehydrated products for a relatively long period of time. Due to anactivity of entrapping a relatively large amount of moisture as high asabout 10% (w/w), α,α-trehalose would be more suitably used than theabove-mentioned other saccharide. The method, however, still needs arelatively large amount of anhydrous α,α-trehalose for dehydration, andtherefore another dehydrating agent having a higher moisture and/ordrying efficiency have been in great demand.

DISCLOSURE OF INVENTION

To overcome the demerits in conventional dehydration methods usingsaccharides, the present inventors have screened natural non-reducingsaccharides in an anhydrous form and energetically studied to establishan improved dehydrating agent and uses thereof.

The present inventors previously established a method for producingcyclotetrasaccharide, which had been known to be only prepared in alaboratory demonstration, at a lesser cost and on an industrial scalefrom material amylaceous saccharides. They also revealed thatcyclotetrasaccharide exists at least in the form of a mono-, penta- orhexa-hydrous crystal as a hydrous crystalline form; or of an anhydrouscrystal or anhydrous amorphous as an anhydrous form. Later, they furtherfound that cyclotetrasaccharide in the form of an anhydrous crystal,monohydrous crystal, or anhydrous amorphous absorbs moisture and easilychanges into its crystalline, penta- or hexa-hydrous form, as a hydrousform.

The present inventors further studied on applying the above features todehydrating agents and resulted in a finding that the above-mentionedcyclotetrasaccharide in the form of an anhydrous crystal, monohydrouscrystal, or anhydrous amorphous, has a satisfactory dehydrating ability;the dehydrated products produced therewith are highly stable. Thus, sucha cyclotetrasaccharide can be widely applicable and more suitably usedas a dehydrating agent as compared with conventional saccharides. Inother words, the present inventors found that a cyclotetrasaccharidewith dehydrating ability, i.e., a saccharide selected fromcyclotetrasaccharides in the form of an anhydrous crystal, monohydrouscrystal, or anhydrous amorphous can be incorporated into, contactedwith, or coexisted in hydrous matters such as hydrous food products andhydrous pharmaceuticals to be converted into crystallinecyclotetrasaccharide, penta- or hexa-hydrate, whereby thecyclotetrasaccharide entraps a relatively large amount of moisture as acrystal water, acts as a dehydrating agent with a remarkably highdehydration power, and has a satisfactory stability. Thus, they foundthat such a cyclotetrasaccharide can be extensively used in hydrousmatters including acid hydrous matters and confirmed that thecyclotetrasaccharide facilitates the production of dehydrated productssuch as high-quality dehydrated food products with satisfactory flavor,and dehydrated pharmaceuticals with satisfactory activity and stability.Thus the present inventors accomplished this invention.

The present invention is characterized in that it was made byappropriately selecting the desired cyclotetrasaccharide which had notbeen focused on its use as a dehydrating agent, particularly, it wasfirstly made by the present invention the method for dehydrating hydrousmatters by incorporating, contacting, or coexisting acyclotetrasaccharide with dehydrating ability into, with, or in hydrousmatters.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an elution pattern of a saccharide, obtained by the enzymaticreaction with α-isomaltosyl-transferring enzyme, when determined onhigh-performance liquid chromatography.

FIG. 2 is a nuclear resonance spectrum (¹H-NMR) of cyclotetrasaccharide,obtained by the enzymatic reaction with α-isomaltosyl-transferringenzyme.

FIG. 3 is a nuclear resonance spectrum (¹³C-NMR) ofcyclotetrasaccharide, obtained by the enzymatic reaction withα-isomaltosyl-transferring enzyme.

FIG. 4 shows that cyclotetrasaccharide has the structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}.

FIG. 5 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C9 strain.

FIG. 6 shows the pH influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C9 strain.

FIG. 7 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C9 strain.

FIG. 8 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus C9strain.

FIG. 9 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain.

FIG. 10 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain.

FIG. 11 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Bacillus globisporus C9 strain.

FIG. 12 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Bacillus globisporus C9 strain.

FIG. 13 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 14 shows the pH influence on α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus C11strain.

FIG. 15 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 16 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus C11strain.

FIG. 17 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C11 strain.

FIG. 18 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C11 strain.

FIG. 19 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Bacillus globisporus C11 strain.

FIG. 20 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Bacillus globisporus C11 strain.

FIG. 21 is a nuclear resonance spectrum (¹H-NMR) ofα-isomaltosylmaltotriose, obtained by the enzymatic reaction withα-isomaltosylglucosaccharide-forming enzyme.

FIG. 22 is a nuclear resonance spectrum (¹H-NMR) ofα-isomaltosylmaltotetraose, obtained by the enzymatic reaction withα-isomaltosylglucosaccharide-forming enzyme.

FIG. 23 is a nuclear resonance spectrum (¹³C-NMR) ofα-isomaltosylmaltotriose, obtained by the enzymatic reaction withα-isomaltosylglucosaccharide-forming enzyme.

FIG. 24 is a nuclear resonance spectrum (¹³C-NMR) ofα-isomaltosylmaltotetraose, obtained by the enzymatic reaction withα-isomaltosylglucosaccharide-forming enzyme.

FIG. 25 is a visualized intermediate picture, displayed on a screen, ofa microscopic photo for crystalline cyclotetrasaccharide, penta- orhexa-hydrate.

FIG. 26 is an x-ray diffraction spectrum for crystallinecyclotetrasaccharide, penta- or hexa-hydrate, when determined on x-raypowder diffraction analysis.

FIG. 27 is a thermogravimetric curve for crystallinecyclotetrasaccharide, penta- or hexa-hydrate, when determined onthermogravimetric analysis.

FIG. 28 is an x-ray diffraction spectrum for crystallinecyclotetrasaccharide, monohydrate, used in the present invention whendetermined on x-ray powder diffraction analysis.

FIG. 29 is a thermogravimetric curve for crystallinecyclotetrasaccharide, monohydrate, used in the present invention whendetermined on thermogravimetric analysis.

FIG. 30 is an x-ray diffraction spectrum for a powder of anhydrouscrystalline cyclotetrasaccharide, obtained by drying in vacuo at 40° C.crystalline cyclotetrasaccharide, penta- or hexa-hydrate, whendetermined on x-ray powder diffraction analysis.

FIG. 31 is an x-ray diffraction spectrum for a powder of anhydrouscrystalline cyclotetrasaccharide, obtained by drying in vacuo at 120° C.cyclotetrasaccharide crystal, penta- or hexa-hydrate, when determined onx-ray powder diffraction analysis.

FIG. 32 is a thermogravimetric curve for an anhydrous crystallinecyclotetrasaccharide powder used in the present invention, whendetermined on thermogravimetric analysis.

FIG. 33 is an x-ray diffraction spectrum for a powder of anhydrouscrystalline cyclotetrasaccharide, obtained by lyophilizing and drying invacuo an aqueous cyclotetrasaccharide solution.

BEST MODE FOR CARRYING OUT THE INVENTION

The method for dehydrating hydrous matters according to the presentinvention is advantageously applied to those which contain water,particularly, to those which contain free water but not bound water suchas a crystal water. For example, the method can be advantageouslyapplied to reduce the water content in the inner atmosphere ofmoisture-proof containers, which hermetically house dried food products,through a step of coexisting the dehydrating agent of the presentinvention therein; or to reduce the free water content of hydrousmatters through a step of incorporating or contacting the dehydratingagent into or with such hydrous matters, for example, food products,cosmetics, pharmaceuticals, industrial chemicals, and their materialsand processing intermediates.

When a cyclotetrasaccharide with dehydrating ability is allowed tocontact with or coexisted in the above hydrous matters, such acyclotetrasaccharide strongly entraps water, as a crystal water ofcrystalline cyclotetrasaccharide, penta- or hexa-hydrate, in an amountof about 11 to 15% (w/w) (the term “% (w/w)” is abbreviated as “%”throughout the specification) to the weight of the cyclotetrasaccharideused, from the hydrous matters, the level of which is 2.2–3-times higherthan that of anhydrous maltose and 1.1–1.5-times higher than that ofanhydrous α,α-trehalose; and effectively lowers the water content of thehydrous matters and dehydrates and/or dries them.

It was revealed that the coexistence of a cyclotetrasaccharide withdehydrating ability, in such a manner of injecting the saccharide intomoisture permeable small bags such as paper bags and placing theresultants in moisture-proof containers which enclose hermetically driedfoods such as seasoned layers and cookies, highly lowers the relativehumidity within the containers and stably keeps the high quality ofdried foods or powdery products for a relatively long period of time. Inthis case, the cyclotetrasaccharide does not either become sticky, meltto flow, or stain the dried foods or the containers even during or afterentrapping water and being converted into crystallinecyclotetrasaccharide, penta- or hexa-hydrate.

As for high moisture content food products, for example, those in theform of a liquid or paste such as brandies, vinegars, royal jellies,fresh creams, and mayonnaises, they can be quite easily processed intohigh quality dehydrated food products having only a substantiallyreduced water content such as food products in the form of a massecuiteor powder, through the steps of incorporating a cyclotetrasaccharidewith dehydrating ability into such high moisture content products toeffect dehydration while the saccharide being converted into itscrystal, penta- or hexa-hydrate.

In that case, when the cyclotetrasaccharide is added to food materialsin an amount sufficient to dehydrate the water in the food materials,the cyclotetrasaccharide is partly converted into crystallinecyclotetrasaccharide, penta- or hexa-hydrate. As a result, the resultingdehydrated food products, where the free water content is reduced, areprevented from quality change and deterioration due to bacterialcontamination, hydrolysis, acidification, or browning; and theirsatisfactory quality, flavor, and taste will be retained for arelatively long period of time.

The dehydrating method of the present invention has the character that,since the cyclotetrasaccharide used in the present invention is anon-reducing saccharide and is free of severe conditions such as heatdrying, high water content products in the form of a liquid or paste canbe easily converted into dehydrated food products with satisfactoryflavor and taste and a reduced water content. Cyclotetrasaccharide perse is a non-toxic and harmless sweetener having a sweetening power ofabout 20% of that of sucrose, and is free of side effect.

In the case of applying the dehydrating method to aqueous solutions oflymphokines or antibiotics and to pasty pharmaceuticals such as ginsengextracts and turtle extracts, they can be quite easily converted intohigh quality dehydrated pharmaceuticals with substantially reduced watercontent, for example, pharmaceuticals in the form of a massecuite orpowder, by incorporating a cyclotetrasaccharide with dehydrating abilityinto the above aqueous solutions and the pasty pharmaceuticals toconvert the cyclotetrasaccharide into its crystal, penta- orhexa-hydrate. According to the dehydrating method, high quality andstable dehydrated pharmaceuticals are prepared because it does notrequire severe conditions such as heat drying and thecyclotetrasaccharide functions as a dehydrating agent and a stabilizerfor the effective ingredients of pharmaceuticals.

For example, solid preparations can be arbitrarily prepared by placingin vials a cyclotetrasaccharide with a sufficiently high level ofdehydrating ability, injecting into the vials an aqueous solutioncontaining a physiologically active substance(s) such as a lymphokine orhormone, and sealing the vials. In this case, the cyclotetrasaccharidedehydrates the aqueous solution and also absorbs/dries the gas spaces ofthe vials. The dehydrated solid pharmaceuticals have the features thatthey are preparable through a relatively easy processing, retain theirhigh quality for a relatively long period of time, and easily dissolvein water in use.

High quality, stable solid preparations can be prepared by mixing aprescribed amount of an aqueous solution containing a physiologicallyactive substance(s) with the cyclotetrasaccharide with a sufficientlyhigh level of dehydrating ability under stirring conditions, anddirectly placing and sealing the resulting power in a container.Further, the solid preparations can be arbitrarily processed in a usualmanner into granules or tablets for use.

Unlike conventionally known dehydrating agents such as a silica gel orcalcium oxide, the dehydrating agent of the present invention,comprising a cyclotetrasaccharide with dehydrating ability, is a non- orlow-caloric saccharide dehydrating agent that is edible andsubstantially non-assimilable when ingested orally, and it can beadvantageously used as a stabilizer for physiologically activesubstances.

The cyclotetrasaccharide and the one with dehydrating ability used inthe present invention should not be restricted to their origins andprocesses. As described later, a cyclotetrasaccharides with dehydratingability, for example, those in an anhydrous crystalline- or anhydrousamorphous-form, can be preferably used because they absorb moisture tobe converted into crystalline cyclotetrasaccharide, penta- orhexa-hydrate. Similarly, crystalline cyclotetrasaccharide, monohydrate,also absorbs moisture to be converted into crystallinecyclotetrasaccharide, penta- or hexa-hydrate, resulting in an exertionof dehydrating action. Accordingly, the cyclotetrasaccharide withdehydrating ability used in the present invention should not be limitedto cyclotetrasaccharide in a completely anhydrous form and includes, forexample, those in a hydrous form as long as they have dehydratingability without any inconvenience. Thus, the cyclotetrasaccharide withdehydrating ability used in the present invention can be defined byevaluating the moisture content of the saccharide using a conventionalmethod such as the Karl Fischer method. Of course, the moisture contentof the cyclotetrasaccharide as the effective ingredient of thedehydrating agent of the present invention should preferably be as lowas possible, desirably, less than 4%, more desirably, less than 3%. Evena cyclotetrasaccharide with a moisture content of 4% or higher but lessthan 10% has dehydrating ability, however, such a saccharide merely hasa relatively lower function and efficiency as a dehydrating agent.

Prior to establishing the present invention, the present inventorsstudied methods for producing cyclotetrasaccharides with dehydratingability, particularly, anhydrous crystalline cyclotetrasaccharide;crystalline cyclotetrasaccharide, monohydrate; and anhydrous amorphouscyclotetrasaccharide.

Methods for producing such cyclotetrasaccharides include, for example,an enzymatic method using amylaceous substances as materials; a methodwhere hydrolytic enzymes, i.e., alternanase is allowed to act onalternan, as disclosed in European Journal of Biochemistry, Vol. 226,pp. 641–648 (1994); a method of converting panose prepared from starchinto cyclotetrasaccharide using α-isomaltosyl-transferring enzyme, asdisclosed in Japanese Patent Application Nos. 229,557/2000 and234,937/2000; and a method of producing cyclotetrasaccharide from starchusing α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme in combination. In addition, asdisclosed in the specifications of the above applications prior to thepresent application, the present inventors revealed that, as a methodfor producing cyclotetrasaccharide, such an enzymatic method usingamylaceous substances which are more abundant and cheaper than alternan,can be advantageously used for industrial scale production because itproduces the desired cyclotetrasaccharide at a relatively highefficiency and a lesser cost. Also they firstly revealed thatcyclotetrasaccharide exists, for example, in the form of a penta- orhexa-hydrate crystal, anhydrous crystal, monohydrous crystal, oranhydrous amorphous crystal.

Examples of microorganisms which formα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme include Bacillus globisporus C9 strainand Bacillus globisporus C11 strain, which were deposited on Apr. 25,2000, and have been maintained in International Patent OrganismDepositary National Institute of Advanced Industrial Science andTechnology Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken, 305-8566, Japan, under the accession numbers of FERMBP-7143 and FERM BP-7144, respectively.

The present inventors further studied the process for producinganhydrous crystalline cyclotetrasaccharide and then established thefollowing; a process comprising the steps of, for example, preparing anaqueous solution of cyclotetrasaccharide, produced from amylaceoussubstances using any of the above-mentioned enzymatic methods, into ahigh concentrated syrup with a concentration of over 2.0% but less than12%, keeping the concentrate at a temperature of 50–180° C. in thepresence of a seed crystal of anhydrous crystallinecyclotetrasaccharide, crystallizing anhydrous crystallinecyclotetrasaccharide, and pulverizing the resulting crystals.

A powdery crystalline cyclotetrasaccharide, monohydrate, is prepared,for example, by adequately drying a powdery cyclotetrasaccharide, penta-or hexa-hydrate, at a temperature of about 100° C. to about 180° C.

To produce anhydrous amorphous cyclotetrasaccharide, for example, anaqueous solution of cyclotetrasaccharide obtained by any of theabove-mentioned methods is lyophilized or dried at a temperature ofabout 100° C. to about 180° C. and at a normal pressure or in vacuo, andpulverizing the resultant. The above aqueous solution ofcyclotetrasaccharide can be prepared into an about 40–85% syrup,lyophilized or dried in vacuo, and pulverized; or can be directlyprocessed into a powder form by the spraying- and the drying-methodssuch as the high pressure nozzle method or the rotatory disk method.

As the method for pulverization, apart from the above spraying anddrying methods, for example, conventional methods such as blockpulverization method, extrusion granulation method, and fluidized-bedgranulation method can be appropriately employed.

The powdery cyclotetrasaccharide with dehydrating ability thus obtainedis a non-reducing, free-flowing, white powder with a high quality, lowsweetness, and is low in moisture content or substantially anhydrous;usually, the moisture content is less than 4%, preferably, less than 3%when determined on the Karl Fisher method. The powder can be made intoan anhydrous crystalline powder, crystalline monohydrate powder, oranhydrous amorphous powder.

Depending on use, the powdery cyclotetrasaccharide with dehydratingability can be arbitrarily sized. In the case of preparing smallportions or tablets such as of medicaments, a cyclotetrasaccharide witha smaller particle size is more preferably used because the smaller theparticle size of cyclotetrasaccharide the more the effective ingredientscan be homogeneously dispersed. The particle size of the dehydratingagent of the present invention can be appropriately controlled byconventional means for classification using meshes, etc.; usually thosewith a particle size of 20–500 μm, preferably, 50–200 μm can bearbitrarily used.

Any powdery cyclotetrasaccharides with dehydrating ability can be usedin the present invention as long as they are anhydrouscyclotetrasaccharides which exert a strong dehydrating action duringtheir conversion into crystalline cyclotetrasaccharide, penta- orhexa-hydrate. For example, preferably used are those which compriseanhydrous cyclotetrasaccharide supplemented with, as a seed, crystallinecyclotetrasaccharide, penta- or hexa-hydrate, in the least possibleamount, usually, in an amount of less than 5%, preferably, less than 1%.

When incorporated into hydrous matters such as food products, cosmetics,pharmaceuticals, and industrial chemicals, the powderycyclotetrasaccharides with dehydrating ability thus obtained act as astrong dehydrating agent for hydrous matters in such a manner ofentrapping and holding the free water in the hydrous matters as acrystal water of crystalline cyclotetrasaccharide, penta- orhexa-hydrate.

Appropriate examples, which the dehydrating agent of the presentinvention is advantageously applicable to, include those wherein theagent is placed in moisture-proof containers to dehydrate or dry theinner atmosphere in the containers, and methods for producinghigh-quality dehydrated products in the form of a massecuite, powder, orsolid by contacting the agent with hydrous matters susceptible to changein quality or deterioration during heat drying or drying in vacuo.

Examples of the above application of the dehydrating agent to dehydrateor dry hydrous matters include those for preventing seasoned layers,cookies, etc., from absorbing moisture. According to the presentinvention, a cyclotetrasaccharide with dehydrating ability can be usedin such a manner of incorporating it into the following powdery productssusceptible to absorbing moisture and solidifying, and then sealing theproducts in containers to lower the relative humidity within thecontainers and prevent the adhesion or solidification of the products,resulting in an exertion of their high quality and satisfactoryfree-flowing ability just after their processings: cereal powders suchas rice powders, wheat flours, and soy bean flours; processed serialssuch as a hattaiko (a flour of heated and ground rice or wheat), kinako(a soy bean powder), and ground sesame; premix flours such as a premixof pudding and hot cake mix; fine granular crystalline seasonings suchas salts and sugars; seasoning powders such as a powdered soy sauce,powdered miso, powdered vinegar for sushi, premix of soup stock powder,and powdered complex seasoning; powdered spices such as a powderedgarlic, powdered cinnamon, powdered nutmeg, powdered pepper, andpowdered sage; and other powdered products such as a powdered yeastextract, powdered milk, powdered yoghurt, powdered cheese, powderedjuice, powdered herb, powdered vitamin, powdered soup, powderedbouillon, fish flour, blood meal, bone meal, powdered preparation oflactic acid bacteria, powdered enzyme preparation, and granulardigestive.

In the case of applying the dehydrating agent of the present inventionto dehydrate hydrous matters, for example, the agent can be arbitrarilyused to dehydrate a variety of hydrous matters such as organs, tissues,and cells of animals, plants, and microorganisms, as well as theirground products, extracts, ingredients, and preparations.

In the case of applying the dehydrating agent of the present inventionto food products and their materials and intermediates, dehydrated foodproducts with satisfactory stability, flavor, and taste can be easilyprocessed from those in the form of a liquid or paste, for example,agricultural products such as a fresh fruit, juice, vegetable extract,soybean milk, sesame paste, nut paste, raw bean's paste, gelatinizedstarch paste, and wheat flour; fishery products such as a paste of seaurchin, oyster extract, and paste of Japanese pilchard; livestockproducts such as a fresh egg, lecithin, milk, milk serum, cream,yoghurt, butter, and cheese; hydrous seasonings such as a maple syrup,honey, miso, soy sauce, mayonnaises, dressing, bonito extract, meatextract, tangle extract, mushroom extract, licorice extract, steviaextract, and their enzyme treated products and seasoning liquids forpickling; alcohols such as a Japanese sake, wine, brandy, whisky, andmedicated liquor; beverages for preference such as a green tea, tea, andcoffee; hydrous spices such as an extract of mint, Japanese horseradish,garlic, mustard, Japanese pepper, cinnamon, sage, laurel, pepper, andcitrus; hydrous colors such as Rubia tinctorum L.; hydrous emulsifiersprepared, for example, from sucrose fatty acid esters, glycerin fattyacid esters, and sorbitan fatty acid esters; and preservation liquidssuch as smoke solutions and fermented liquids.

Among the dehydrated products thus obtained, the powdered products ofagriculture, fishery, and livestock; powdered flavors; powdered colors;powdered emulsifiers; and powdered preservatives can be arbitrarily usedas natural bulk flavors with satisfactory flavor and taste or processingmaterials in seasonings such as mayonnaises and soup premixes,confectioneries such as hard candies and cakes, and premixes such as hotcake mixes and instant juices.

In the case of applying the dehydrating agent of the present inventionto pharmaceuticals and their materials and processing intermediates,dehydrated pharmaceuticals and health foods with satisfactory stabilityand high quality without losing the effective ingredients and activitiesin the following liquid or paste products: Examples of such are liquidscontaining lymphokines such as α-, β- or γ-interferon, tumor necrosisfactor-α (TNF-α), tumor necrosis factor-β (TNF-β), macrophage migrationinhibitory factor, colony-stimulating factor, transfer factor andinterleukin 2; liquids containing hormones such as insulin, growthhormone, prolactin, erythropoietin, and follicle-stimulating hormone;liquids containing biological preparations such as BCG vaccine, Japaneseencephalitis vaccine, measles vaccine, live polio vaccine, smallpoxvaccine, tetanus toxoid, Trimeresurus antitoxin, and humanimmunoglobulin; liquids containing antibiotics such as penicillin,erythromycin, chloramphenicol, tetracycline, streptomycin, and kanamycinsulfate; liquids containing vitamins such as thiamine, riboflavin,L-ascorbic acid, cod liver oil, carotenoid, ergosterol, and tocopherol;liquids containing enzymes such as lipase, elastase, urokinase,protease, β-amylase, isoamylase, glucanase, and lactase; extracts suchas ginseng extract, snapping turtle extract, chlorella extract, aloeextract, and propolis extract; pastes of viable microorganisms such asviruses, lactic acid bacteria, and yeasts; and other liquid or pasteproducts such as royal jellies.

Similarly as in the above food products and pharmaceuticals, thedehydrating agent of the present invention is applied to cosmetics andtheir materials and processing intermediates such as fresh eggs,lecithin, creams, honeys, licorice extracts, flavors, colors, andenzymes to dehydrate them into high quality, dehydrated cosmetics whichare advantageously used as skin-beautifying agents, hair-beautifyingagents, and hair restorers.

In the case of applying the dehydrating agent to enzyme preparations tobe dehydrated, the obtained preparations can be arbitrarily used ascatalysts for processing food products, pharmaceuticals, and industrialmaterials; or as therapeutic agents, digestives, or enzyme detergents.

The methods for incorporating, contacting or coexisting thecyclotetrasaccharide with dehydrating ability into, with, or in hydrousmatters are mixing, kneading, dissolving, penetrating, dispersing,applying, spraying, and injecting, which are appropriately selectedbefore completing the dehydration of the hydrous matters.

Depending on the water content of hydrous matters to be dehydrated andthe properties of the desired dehydrated matters, it is varied theamount of a cyclotetrasaccharide with dehydrating ability to beincorporated into, or contacted with or coexisted in hydrous matters. Ifnecessary, prior to incorporate, contact or coexist thecyclotetrasaccharide with dehydrating ability into, with, or in hydrousmatters, the hydrous matters are preferably partially dehydrated orconcentrated with other conventional dehydrating methods. Usually,0.001–200 parts by weight, preferably, 0.01–50 parts by weight of thecyclotetrasaccharide is used to one part by weight of a hydrous matter.

To improve the quality of the resulting dehydrated products such as foodproducts, cosmetics, and pharmaceuticals, appropriate flavors, colors,taste-imparting materials, stabilizers, and fillers can be arbitrarilyused in combination. Particularly, since the dehydrating method of thepresent invention is a quite effective dehydration method using acyclotetrasaccharide with strong dehydrating ability, the aforesaidstabilizers should not be limited to lower molecular weight compoundssuch as antioxidants. The following water-soluble high molecular weightcompounds, whose dehydration have been deemed difficult, can bearbitrarily used as such stabilizers; soluble starch, dextrin, pullulan,elsinan, dextran, xanthan gum, gum arabic, locust bean gum, guar gum,tragacanth gum, tamarind gum, carboxy methyl cellulose, hydroxy ethylcellulose, pectin, agar, gelatin, albumin, and casein.

In the case of using the above water-soluble high molecular compounds,for example, they are first homogeneously dissolved in hydrous mattersin a liquid or paste form, then a cyclotetrasaccharide with dehydratingability is incorporated into the resulting mixtures by the methods suchas mixing and kneading to obtain dehydrated products with finecyclotetrasaccharide crystals, penta- or hexa-hydrate.

The above dehydrated matters quite advantageously, stably retain theflavor and effective ingredients from the hydrous matters because theingredients are prevented from dispersion and deterioration by eitherthe coating with films of the high molecular substances; theencapsulation in microcapsules, surrounded with the films, together withthe fine cyclotetrasaccharide crystals, penta- or hexa-hydrate; or thestabilization of flavor ingredients or effective ingredients by forminginclusion compounds using a cyclotetrasaccharide with dehydratingability. In this case, if necessary, α-, β- or γ-cyclodextrin, capableof forming inclusion compounds of flavor ingredients, can be arbitrarilyused as the water-soluble high molecular substances.

The cyclodextrins usable in the present invention should not be limitedto those with a high purity but arbitrarily include those with a lowpurity, which are hard to be dehydrated and pulverized, for example,glucose derivatives of cyclodextrins and partial starch hydrolyzates inthe form of a hydrolyzed starch syrup rich in maltodextrins andcontaining different types of cyclodextrins.

The methods used for producing the dehydrated products according to thepresent invention, particularly, those for pulverized dehydratedproducts are variable. For example, a cyclotetrasaccharide withdehydrating ability is incorporated to homogeneity into hydrous matterswith a relatively high moisture content such as food products,cosmetics, pharmaceuticals, and their materials and processingintermediates to give a moisture content of about 50% or lower,preferably, about 10 to 40% to the total weight of the resultingdehydrated products. The resulting mixtures are then allowed to stand ina vat at a temperature of about 10° C. to about 50° C., for example, atambient temperature, for about 0.1 to about 5 days to be solidified intoblocks through the conversion of the cyclotetrasaccharide intocrystalline cyclotetrasaccharide, penta- or hexa-hydrate, followed bycutting and pulverizing the blocks. If necessary, drying and classifyingsteps can follow a pulverization step such as cutting and pulverizing.

By applying a spraying method, pulverized products can be directlyproduced. For example, the methods below can be suitably used as anindustrial scale production method of the powdery dehydrated products ofthe present invention: A method comprising the steps of spraying aprescribed amount of a liquid or paste hydrous matter over acyclotetrasaccharide with dehydrating ability under free-flowingconditions to contact them each other, granulating the resultingmixture, and optionally aging the resulting granules at about 30° C. toabout 60° C. for about 0.1 to about 10 hours to convert thecyclotetrasaccharide into crystalline cyclotetrasaccharide, penta- orhexa-hydrate; or a method comprising the steps of mixing and kneading aliquid or pasty hydrous matter with a cyclotetrasaccharide withdehydrating ability, and then instantly or after initiating theconversion of the cyclotetrasaccharide into crystallinecyclotetrasaccharide, penta- or hexa-hydrate, and optionally sprayingthe resultant into a powder and optionally aging similarly as above toconvert the remaining cyclotetrasaccharide into crystallinecyclotetrasaccharide, penta- or hexa-hydrate.

The powdery dehydrated products thus obtained can be arbitrarily usedintact or, if necessary, in combination with fillers, adjuvants,binders, stabilizers, etc., or after processed into an appropriate formsuch as a granule, tablet, capsule, rod, plate, or cubic.

Since starch generally requires a relatively large amount of water forswelling and gelatinizing, the resulting swelled and gelatinized starchis highly susceptible to bacterial contamination. Thecyclotetrasaccharide with dehydrating ability can be effectively used asa dehydrating agent for such gelatinized starch. For example,gelatinized starch such as “gyuhi” (a rice paste with sugar) can beprevented from bacterial contamination by incorporating acyclotetrasaccharide with dehydrating ability thereunto to substantiallyreduce the water content thereof.

The cyclotetrasaccharide with dehydrating ability can be easily mixed tohomogeneity with gelatinized starch and acts as a retrogradationpreventive as described later, and it can prolong the shelf-life ofprocessed foods containing gelatinized starch by a large margin.

In applying a cyclotetrasaccharide with dehydrating ability over thesurface of high moisture content food products, which are susceptible tobacterial contamination, such as a peeled banana, orange, slicedsteamed/boiled sweet-potato, split jack, hairtail, raw noodle, boilednoodle, and rice confectionery, the cyclotetrasaccharide contacts thefood products by sprinkling over the surface of the food products toconvert the cyclotetrasaccharide into crystalline cyclotetrasaccharide,penta- or hexa-hydrate, resulting in a substantial reduction of thewater content on the surface of the food products and an improvement oftheir shelf-life and quality. For this reason the cyclotetrasaccharidewith dehydrating ability can be advantageously used as a foodpreservative, stabilizer, or quality-improving agent. In this case, theshelf-life of the above food products can be further prolonged byoptionally using lactic acid, citric acid, or ethanol; or by vacuumpackage, gas-filling package, or refrigeration.

Since the cyclotetrasaccharide with dehydrating ability has a relativelyhigh affinity to alcohols, it can be arbitrarily used as a dehydratingagent for water contained in alcohols and alcohol-soluble products suchas methanol, ethanol, butanol, propylene glycol, glycerine, andpolyethylene glycol.

For example, alcohols such as sake, shochu (a distilled spirit), wine,brandy, whisky, and vodka can be advantageously processed into amassecuite or powder which retains the effective ingredients and flavorsof these alcohols by dehydrating the alcohols using acyclotetrasaccharide with dehydrating ability to form crystallinecyclotetrasaccharide, penta- or hexa-hydrate, while allowing toincorporate their effective ingredients and flavors into the crystal.The powdery alcohols thus obtained can be used in confectioneries andpremixes which are mixed with water into beverages before use.

When coexisted in dehydrated hydrous matters, the cyclotetrasaccharidewith dehydrating ability used in the present invention functions as adehydrating agent or stabilizer and also exerts an effect as an agentfor imparting high-quality sweetness, body, or adequate viscosity.

By mixing a cyclotetrasaccharide with dehydrating ability with analcohol solution such as of iodine and then with an aqueous solutioncontaining a water-soluble high molecule, etc., the cyclotetrasaccharideis converted into its crystal, penta- or hexa-hydrate, resulting instabilizing the effective ingredients such as iodine withoutvolatilizing and deteriorating them, and facilitating the production ofointments in a massecuite form having an adequate viscosity,extendibility, and adhesion.

Food products such as powdery fats and oils, spices, flavors, and foodcolors; cosmetics; and pharmaceuticals such as powdery vitamins andhormones are advantageously obtainable by soaking or mixingwater-containing-oil-soluble substances, emulsions, or latexes in orwith the cyclotetrasaccharide with dehydrating ability to convert acyclotetrasaccharide with dehydrating ability into its crystal, penta-or hexa-hydrate.

In such a case, the cyclotetrasaccharide with dehydrating abilityfunctions as a dehydrating agent and as a stabilizer, property-retainingagent, filler, or carrier even after converted into cyclotetrasaccharidecrystal, penta- or hexa-hydrate.

The cyclotetrasaccharide with dehydrating ability used in the presentinvention can be advantageously used even in food products containingoil-soluble substances, which are incompatible with water, such aschocolates and creams. In this case, the cyclotetrasaccharide is usednot only as a dehydrating agent but used for improving processibility,meltability in mouth, flavor and taste. The products thus obtained havea feature of stably retaining their high quality for a relatively longperiod of time.

As described above, the present invention was made based on the findingthat a cyclotetrasaccharide with dehydrating ability strongly absorbsmoisture from hydrous matters, and that the resulting dehydratedproducts are highly stable. When used as a dehydrating agent, thecyclotetrasaccharide with dehydrating ability dehydrates hydrous mattersin a liquid or paste form and facilitates to produce high qualitypharmaceuticals and cosmetics with reduced moisture content withoutdeteriorating or volatilizing the taste or the flavor of the hydrousmatters by the characteristic enclosing action of thecyclotetrasaccharide.

The following are the preferred examples for use according to thepresent invention:

The cyclotetrasaccharide with dehydrating ability used in the presentinvention has a relatively low sweetness and can be also used as aseasoning free from caries inducibility and increment of bloodcholesterol- and/or blood sugar-levels. If necessary, thecyclotetrasaccharide can be used, for example, by mixing with othersweetener(s) such as a powdered syrup, glucose, isomerized sugar,sucrose, maltose, α,α-trehalose, honey, maple sugar, sorbitol, maltitol,dihydrocharcone, stevioside, α-glycosyl stevioside, rebaudioside,glycyrrhizin, thaumatin, L-aspartyl L-phenylalanine methyl ester,acesulfame K, sucralose, saccharin, glycine, and alanine; and fillerssuch as dextrin, starch, and lactose.

The cyclotetrasaccharide with dehydrating ability is a non-reducingsaccharide which has a high quality sweetness inherent tocyclotetrasaccharide; well harmonizes with other tastable materialshaving sour-, acid-, salty-, astringent-, delicious-tastes, andbitter-tastes; and has a relatively high acid- and heat-resistance.Thus, the cyclotetrasaccharide can be favorably used in food products ingeneral as a sweetener, taste-improving agent, quality-improving agent,or flavor-improving agent.

The cyclotetrasaccharide can be used as a dehydrating agent inseasonings such as a soy sauce, powdered soy sauce, “miso”,“funmatsu-miso” (a powdered miso), “moromi” (a refined sake), “hishio”(a refined soy sauce), “furikake” (a seasoned fish meal), mayonnaise,dressing, vinegar, “sanbai-zu” (a sauce of sugar, soy sauce andvinegar), “funmatsu-sushi-su” (powdered vinegar for sushi),“chuka-no-moto” (an instant mix for Chinese dish), “tentsuyu” (a saucefor Japanese deep-fat fried food), “mentsuyu” (a sauce for Japanesevermicelli), sauce, catsup, “yakiniku-no-tare” (a sauce for Japanesegrilled meat), curry roux, instant stew mix, instant soup mix,“dashi-no-moto” (an instant stock mix), “mirin” (a sweet sake),“shin-mirin” (a synthetic mirin), table sugar, and coffee sugar. Also,the cyclotetrasaccharide can be arbitrarily used as a sweetener,taste-improving agent, quality-improving agent, ortaste/flavor-improving agent. Further, the cyclotetrasaccharide can befreely used as a dehydrating agent, sweetener, taste-improving agent,quality-improving agent, or taste/flavor-improving agent in “wagashi”(Japanese cakes) such as “senbei” (a rice cracker), “arare-mochi” (arice-cake cube), “okoshi” (a millet-and-rice cake), “gyuhi” (a ricepaste with sugar), “mochi” (a rice paste) or the like, “manju” (a bunwith a bean-jam), “uiro” (a sweet rice jelly), “an” (a bean jam) or thelike, “yokan” (a sweet jelly of beans), “mizu-yokan” (a soft adzuki-beanjelly), “kingyoku” (a kind of yokan), jelly, pao de Castella, and“amedama” (a Japanese toffee); confectioneries such as a bun, biscuit,cracker, cookie, pie, pudding, butter cream, custard cream, cream puff,waffle, sponge cake, doughnut, chocolate, chewing gum, caramel, nougat,and candy; frozen desserts such as an ice cream and sherbet; syrups suchas “kajitsu-no-syrup-zuke” (a preserved fruit) and “korimitsu” (a sugarsyrup for shaved ice); pastes such as a flour paste, peanut paste, andfruit paste; processed fruits and vegetables such as a jam, marmalade,“syrup-zuke” (fruit pickles), and “toka” (conserves); pickles andpickled products such as “fukujin-zuke” (red colored radish pickles),“bettara-zuke” (a kind of whole fresh radish pickles), “senmai-zuke” (akind of sliced fresh radish pickles) and “rakkyo-zuke” (pickledshallots); premixes for pickles and pickled products such as“takuan-zuke-no-moto” (a premix for pickled radish) and“hakusai-zuke-no-moto” (a premix for fresh white rape pickles); meatproducts such as a ham and sausage; products of fish meat such as a fishham, fish sausage, “kamaboko” (a steamed fish paste), “chikuwa” (a kindof fish paste), and “tenpura” (a Japanese deep-fat fried fish paste);“chinmi” (relishes) such as “uni-no-shiokara” (salted guts of seaurchin), “ika-no-shiokara” (salted guts of squid), “su-konbu” (processedtangle), “saki-surume” (dried squid strips) and “fugu-no-mirin-boshi” (adried mirin-seasoned swellfish); “denpu” (a fish meet boiled down,seasoned, and dried) such as those of cod, sea bream, and shrimp;“tsukudani” (foods boiled down in soy sauce) such as those of layer,edible wild plants, dried squid, fish, and shellfish; daily dishes suchas “nimame” (a cooked bean), potato salad, and “konbu-maki” (a tangleroll); milk products; canned and bottled products such as those of meat,fish meat, fruit, and vegetable; alcoholic beverages such as a syntheticsake, wine, and liquors; soft drinks such as coffee, tea, cocoa, juice,carbonated beverage, sour milk beverage, and beverage containing lacticacid bacteria; and instant food products such as an instant pudding mix,instant hot cake mix, “sokuseki-shiruco” (an instant mix of adzuki-beansoup with rice cake), and instant soup mix.

The following explain the process for producing thecyclotetrasaccharides usable in the present invention and propertiesthereof:

Experiment 1

Preparation of Cyclotetrasaccharide from Culture

A liquid culture medium consisting of 5% (w/v) of “PINE-DEX #1”, apartial starch hydrolysate commercialized by Matsutani Chemical Ind.,Tokyo, Japan, 1.5% (w/v) of “ASAHIMEAST”, a yeast extract commercializedby Asahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) of sodium dihydrogenphosphate, dodecahydrate, 0.06% (w/v) of magnesium sulfate,dodecahydrate, and water was placed in a 500-ml Erlenmeyer flask in anamount of 100 ml, sterilized by autoclaving at 121° C. for 20 min,cooled, and then seeded with Bacillus globisporus C9 strain, FERMBP-7143, followed by culturing under rotary-shaking conditions at 27° C.and 230 rpm for 48 hours and centrifuging the resulting culture toremove cells to obtain a supernatant. The supernatant was autoclaved at120° C. for 15 min and then cooled, and the resulting insolublesubstances were removed by centrifugation to obtain a supernatant.

To examine the saccharides in the resulting supernatant, they wereseparated from the supernatant on silica gel thin-layer chromatography(abbreviated as “TLC” hereinafter) using, as a developer, a mixturesolution of n-butanol, pyridine, and water (=6:4:1 by volume), and, as athin-layer plate, “KIESELGEL 60”, an aluminum plate (20×20 cm) for TLCcommercialized by Merck & Co., Inc., Rahway, USA. Whole of the separatedsugars and the reducing sugars among them were respectively examined bycoloring with the sulfuric acid-methanol method and thediphenylamine-aniline method. The examination detected a non-reducingsaccharide, positively detected on the former detection method at aposition with an Rf value of about 0.31 but negative on the latterdetection method.

About 90 ml of the above supernatant was adjusted to pH 5.0 and 45° C.and then treated for 24 hours with 1,500 units/g solids of“TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase commercialized by AmanoPharmaceutical Co., Ltd., Aichi, Japan, and 75 units/g solids of aglucoamylase commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan.Then, the resulting mixture was adjusted to pH 12 by the addition ofsodium hydroxide and boiled for two hours to decompose the remainingreducing sugars. After removing insoluble substances by filtration, theresulting solution was decolored and desalted with “DIAION PK218” and“DIAION WA30”, cation exchange resins commercialized by MitsubishiChemical Industries, Ltd., Tokyo, Japan, and further desalted with“DIAION SK-1B”, commercialized by Mitsubishi Chemical Industries, Ltd.,Tokyo, Japan, and “AMBERLITE IRA411”, an anion exchange resincommercialized by Japan Organo Co., Ltd., Tokyo, Japan, followed bysuccessive decoloration with an activated charcoal, membrane filtration,concentration by an evaporator, and lyophilization in vacuo to obtainabout 0.6 g, d.s.b., of a saccharide powder.

The analysis of the saccharide on high-performance liquid chromatography(abbreviated as “HPLC” hereinafter) detected only a single peak at anelution time of 10.84 min as shown in FIG. 1, revealing that thesaccharide had a significantly high purity of 99.9% or higher. The HPLCwas run using a column of “SHOWDEX KS-801”, Showa Denko K.K., Tokyo,Japan, at a column temperature of 60° C. and a flow rate of 0.5 ml/minof water, and using “R1-8012”, a differential refractometercommercialized by Tosoh Corporation, Tokyo, Japan.

When measured for reducing power on the Somogyi-Nelson's method, thespecimen had a reducing power below a detectable level, revealing thatthe specimen was a substantially non-reducing saccharide.

Experiment 2

Structure Analysis of Cyclotetrasaccharide

Fast atom bombardment mass spectrometry (called “FAB-MS”) on anon-reducing saccharide, obtained by the method in Experiment 1, clearlydetected a proton-addition-molecular ion with a mass number of 649,revealing that the saccharide had a mass number of 648.

According to conventional manner, the saccharide was hydrolyzed withsulfuric acid and analyzed for sugar composition on gas chromatography.As a result, only D-glucose was detected, revealing that the saccharidetested was composed of D-glucose molecules. Based on the data and theabove mass number, the saccharide was estimated to be acyclotetrasaccharide, composed of four D-glucose molecules.

Nuclear magnetic resonance analysis (called “NMR”) on the saccharidegave a ¹H-NMR spectrum in FIG. 2 and a ¹³C-NMR spectrum in FIG. 3, andthese spectra were compared with those of authentic saccharides,revealing that they were coincided with a non-reducing cyclicsaccharide,cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}as disclosed in “European Journal of Biochemistry”, pp. 641–648 (1994).The data confirmed that the saccharide was a cyclotetrasaccharide inFIG. 4, i.e.,cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}.

Experiment 3

Production of α-Isomaltosylglucosaccharide-Forming Enzyme from Bacillusglobisporus C9 Strain

A liquid culture medium consisting of 4.0% (w/v) of “PINE-DEX #4”, apartial starch hydrolysate commercialized by Matsutani Chemical Ind.,Tokyo, Japan, 1.8% (w/v) of “ASAHIMEAST”, a yeast extract commercializedby Asahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassiumphosphate, 0.06% (w/v) of sodium dihydrogen phosphate, dodecahydrate,0.05% (w/v) magnesium sulfate, heptahydrate, and water was placed in500-ml Erlenmeyer flasks respectively in an amount of 100 ml, sterilizedby autoclaving at 121° C. for 20 min, cooled, and then seeded with astock culture of Bacillus globisporus C9 strain, FERM BP-7143, followedby culturing under rotary-shaking conditions at 27° C. and 230 rpm for48 hours for a seed culture.

About 20 L of a fresh preparation of the same liquid culture medium asused in the above seed culture was placed in a 30-L fermentor,sterilized by heating, and then cooled to 27° C. and inoculated with 1%(v/v) of the seed culture, followed by culturing at 27° C. and a pH of6.0 to 8.0 for 48 hours under aeration-agitation conditions. Aftercompletion of the culture, the resulting culture, which had about 0.45unit/ml of α-isomaltosylglucosaccharide-forming enzyme activity, about1.5 units/ml of α-isomaltosyl-transferring enzyme activity, and about0.95 unit/ml of cyclotetrasaccharide-forming enzyme activity, wascentrifuged at 10,000 rpm for 30 min to obtain about 18 L of asupernatant. When measured for enzymatic activity, the supernatant hadan activity of about 0.45 unit/ml ofα-isomaltosylglucosaccharide-forming enzyme, i.e., a total enzymeactivity of about 8,110 units; about 1.5 units/ml ofα-isomaltosyl-transferring enzyme, i.e., a total enzyme activity ofabout 26,900 units; and about 0.95 unit/ml ofcyclotetrasaccharide-forming activity, i.e., a total enzyme activity ofabout 17,100 units.

The activities of these enzymes were assayed as follows: The activity ofα-isomaltosylglucosaccharide-forming enzyme was assayed by dissolvingmaltotriose in 100 mM acetate buffer (pH 6.0) to give a concentration of2% (w/v) for a substrate solution, adding 0.5 ml of an enzyme solutionto 0.5 ml of the substrate solution, enzymatically reacting the mixturesolution at 35° C. for 60 min, boiling the reaction mixture for 10 minto suspend the enzymatic reaction, and quantifying maltose, among theisomaltosyl maltose and maltose formed in the reaction mixture, on HPLCas described in Experiment 1. One unit activity of theα-isomaltosylglucosaccharide-forming enzyme was defined as the enzymeamount that forms one micromole of maltose per minute under the aboveenzymatic reaction conditions.

The activity of α-isomaltosyl-transferring enzyme was assayed bydissolving panose in 100 mM acetate buffer (pH 6.0) to give aconcentration of 2% (w/v) for a substrate solution, adding 0.5 ml of anenzyme solution to 0.5 ml of the substrate solution, enzymaticallyreacting the mixture solution at 35° C. for 30 min, boiling the reactionmixture for 10 min to suspend the enzymatic reaction, and quantifyingglucose, among the cyclotetrasaccharide and glucose mainly formed in thereaction mixture, by the glucose oxidase method. One unit activity ofthe α-isomaltosyl-transferring enzyme was defined as the enzyme amountthat forms one micromole of glucose per minute under the above enzymaticreaction conditions.

The cyclotetrasaccharide-forming activity was assayed by dissolving“PINE-DEX #100”, a partial starch hydrolysate commercialized byMatsutani Chemical Ind., Tokyo, Japan, in 50 mM acetate buffer (pH 6.0)to give a concentration of 2% (w/v) for a substrate solution, adding 0.5ml of an enzyme solution to 0.5 ml of the substrate solution,enzymatically reacting the mixture solution at 35° C. for 60 min,boiling the reaction mixture at 100° C. for 10 min to suspend theenzymatic reaction, and then further adding to the resulting mixture onemilliliter of 50 mM acetate buffer (pH 5.0) with 70 units/ml of“TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase commercialized by AmanoPharmaceutical Co., Ltd., Aichi, Japan, and 27 units/ml of glucoamylasecommercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, incubatingthe mixture at 50° C. for 60 min, inactivating the remaining enzymes byheating at 100° C. for 10 min, and quantifying cyclotetrasaccharide onHPLC described in Experiment 1. One unit of cyclotetrasaccharide-formingactivity was defined as the enzyme amount that forms one micromole ofcyclotetrasaccharide per minute under the above enzymatic reactionconditions.

Experiment 4

Preparation of Enzyme from Bacillus globisporus C9 Strain

Experiment 4-1

Purification of Enzyme from Bacillus globisporus C9 Strain

About 18 L of the supernatant in Experiment 3 was salted out under 80%saturated ammonium sulfate and allowed to stand at 4° C. for 24 hours,and the formed sediments were collected by centrifugation at 10,000 rpmfor 30 min, dissolved in 10 mM phosphate buffer (pH 7.5), and dialyzedagainst a fresh preparation of the same buffer to obtain about 400 ml ofa crude enzyme solution with 8,110 units of theα-isomaltosylglucosaccharide-forming enzyme, 24,700 units ofα-isomaltosyl-transferring enzyme, and about 15,600 units ofcyclotetrasaccharide-forming activity. The crude enzyme solution wassubjected to ion-exchange chromatography using 1,000 ml of “SEPABEADSFP-DA13” gel, an ion-exchange resin commercialized by MitsubishiChemical Industries, Ltd., Tokyo, Japan.α-Isomaltosylglucosaccharide-forming enzyme and cyclotetrasaccharidewere eluted as non-adsorbed fractions without adsorbing on the gel. Theresulting enzyme solution was dialyzed against 10 mM phosphate buffer(pH 7.0) with 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove impurities, and subjected to affinitychromatography using 500 ml of “SEPHACRYL HR S-200”, a gelcommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA. Enzymatically active components adsorbed on the geland, when sequentially eluted with a linear gradient decreasing from 1 Mto 0 M of ammonium sulfate and a linear gradient increasing from 0 mM to100 mM of maltotetraose, α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme were separately eluted, i.e., theformer was eluted with the linear gradient of maltotetraose at about 30mM and the latter was eluted with the linear gradient of ammoniumsulfate at about 0 M. Fractions with α-isomaltosyl-transferring activityand those with α-isomaltosylglucosaccharide-forming activity wereseparatory collected. No cyclotetrasaccharide-forming activity was foundin any of the above fractions and this revealed that a mixture solutionof both of the above fractions with α-isomaltosylglucosaccharide-formingenzyme and α-isomaltosyl-transferring enzyme exhibitedcyclotetrasaccharide-forming activity, showing that the activity offorming cyclotetrasaccharide from partial starch hydrolyzates wasexerted by the coaction of the activities of the above two types ofenzymes.

Methods for separatory purifying α-isomaltosylglucosaccharide-formingenzyme and α-isomaltosyl-transferring enzyme are explained below:

Experiment 4-2

Purification of α-Isomaltosylglucosaccharide-Forming Enzyme

A fraction of the α-isomaltosylglucosaccharide-forming enzyme, obtainedin Experiment 4-1, was dialyzed against 10 mM phosphate buffer (pH 7.0)with 1 M ammonium sulfate. The dialyzed solution was centrifuged toremove insoluble substances, and the resulting supernatant was fed tohydrophobic chromatography using 350 ml of “BUTYL-TOYOPEARL 650 M”, agel commercialized by Tosoh Corporation, Tokyo, Japan. The enzymeadsorbed on the gel and was eluted therefrom at about 0.3 M ammoniumsulfate with a linear gradient decreasing from 1 M to 0 M of ammoniumsulfate, followed by collecting fractions with the enzyme activity. Thefractions were pooled and dialyzed against 10 mM phosphate buffer (pH7.0) with 1 M ammonium sulfate. The resulting dialyzed solution wascentrifuged to remove insoluble substances and fed to affinitychromatography using “SEPHACRYL HR S-200” gel to purify the enzyme. Theamount of enzyme activity, the specific activity, and the yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare in Table 1.

TABLE 1 Specific activity Enzyme* activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 8,110 0.12 100Dialyzed solution after 7,450 0.56 91.9 salting out with ammoniumsulfate Eluate from ion-exchange 5,850 1.03 72.1 column chromatographyEluate from affinity 4,040 8.72 49.8 column chromatography Eluate fromhydrophobic 3,070 10.6 37.8 column chromatography Eluate from affinity1,870 13.6 23.1 column chromatography Note: The symbol “*” meansα-isomaltosylglucosaccharide-forming enzyme.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, meaning a high purity enzyme specimen.

Experiment 4-3

Purification of α-Isomaltosyl-Transferring Enzyme

A fraction with α-isomaltosyl-transferring enzyme, which had beenseparated from a fraction with α-isomaltosylglucosaccharide-formingenzyme by affinity chromatography in Experiment 4-1, was dialyzedagainst 10 mM phosphate buffer (pH 7.0) with 1 M ammonium sulfate. Theresulting dialyzed solution was centrifuged to remove insolublesubstances and subjected to affinity chromatography using 350 ml of“BUTYL-TOYOPEARL 650 M”, a gel commercialized by Tosoh Corporation,Tokyo, Japan. The enzyme adsorbed on the gel and was eluted therefrom atabout 0.3 M ammonium sulfate with a linear gradient decreasing from 1 Mto 0 M of ammonium sulfate, followed by collecting fractions with theenzyme activity. The fractions were pooled and dialyzed against 10 mMphosphate buffer (pH 7.0) with 1 M ammonium sulfate. The resultingdialyzed solution was centrifuged to remove insoluble substances and fedto affinity chromatography using “SEPHACRYL HR S-200” gel to purify theenzyme. The amount of enzyme activity, the specific activity, and theyield of α-isomaltosyl-transferring enzyme in each purification step arein Table 2.

TABLE 2 Specific activity Enzyme* activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 26,900 0.41 100Dialyzed solution after 24,700 1.85 91.8 salting out with ammoniumsulfate Eluate from ion-exchange 19,400 3.41 72.1 column chromatographyEluate from affinity 13,400 18.6 49.8 column chromatography Eluate fromhydrophobic 10,000 21.3 37.2 column chromatography Eluate from affinity6,460 26.9 24.0 column chromatography Note: The symbol “*” meansα-isomaltosyl-transferring enzyme.Experiment 5Property of α-Isomaltosylglucosaccharide-Forming Enzyme andα-Isomaltosyl-Transferring EnzymeExperiment 5-1Property of α-Isomaltosylglucosaccharide-Forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 4-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight in comparison with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Bio-RadLaboratories Inc., Brussels, Belgium, revealing that the enzyme had amolecular weight of about 140,000±20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 5.2±0.5.

The influence of temperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for the enzyme activity, where the influence oftemperature was conducted in the presence or absence of 1 mM Ca²⁺. Theseresults are in FIG. 5 (influence of temperature) and FIG. 6 (influenceof pH). The optimum temperature of the enzyme was about 40° C. (in theabsence of Ca²⁺) and about 45° C. (in the presence of 1 mM Ca²⁺) whenincubated at pH 6.0 for 60 min, and the optimum pH of the enzyme wasabout 6.0 to about 6.5 when incubated at 35° C. for 60 min. The thermalstability of the enzyme was determined by incubating it in 20 mM acetatebuffers (pH 6.0) at prescribed temperatures for 60 min in the presenceor absence of 1 mM Ca²⁺, cooling the resulting enzyme solutions withwater, and assaying the remaining enzyme activity for each solution. ThepH stability of the enzyme was determined by keeping it in 50 mM buffershaving prescribed pHs at 4° C. for 24 hours, adjusting the pH of eachsolution to 6.0, and assaying the remaining enzyme activity for eachsolution. These results are respectively in FIG. 7 (thermal stability)and FIG. 8 (pH stability). As a result, the enzyme was thermally stableup to about 35° C. in the absence of Ca²⁺ and about 40° C. in thepresence of 1 mM Ca²⁺, and was stable at pHs from about 4.5 to about9.0.

The influence of metal ions on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in the presenceof 1 mM of any of metal salts according to the assay for the enzymeactivity. The results are in Table 3.

TABLE 3 Metal Relative activity Metal Relative activity ion (%) ion (%)None 100 Hg²⁺ 4 Zn²⁺ 92 Ba²⁺ 65 Mg²⁺ 100 Sr²⁺ 80 Ca²⁺ 115 Pb²⁺ 103 Co²⁺100 Fe²⁺ 98 Cu²⁺ 15 Fe³⁺ 97 Ni²⁺ 98 Mn²⁺ 111 Al³⁺ 99 EDTA 20

As evident form the results in Table 3, the enzyme activity wassignificantly inhibited by Hg²⁺, Cu²⁺, and EDTA, and was also inhibitedby Ba²⁺ and Sr²⁺. It was also found that the enzyme was activated byCa²⁺ and Mn²⁺.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:1, i.e.,tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucinein the N-terminal region.

Experiment 5-2

Property of α-Isomaltosyl-Transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 4-3, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight incomparison with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Bio-Rad LaboratoriesInc., Brussels, Belgium, revealing that the enzyme had a molecularweight of about 112,000±20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 5.5±0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for the enzyme activity. These results are respectively in FIG. 9(influence of temperature) and FIG. 10 (influence of pH). The optimumtemperature of the enzyme was about 45° C. when incubated at pH 6.0 for30 min, and the optimum pH of the enzyme was about 6.0 when incubated at35° C. for 30 min. The thermal stability of the enzyme was determined byincubating it in 20 mM acetate buffers (pH 6.0) at prescribedtemperatures for 60 min, cooling the resulting enzyme solutions withwater, and assaying the remaining enzyme activity of each solution. ThepH stability of the enzyme was determined by keeping it in 50 mM buffershaving prescribed pHs at 4° C. for 24 hours, adjusting the pH of eachsolution to 6.0, and assaying the remaining enzyme activity for eachsolution. These results are respectively in FIG. 11 (thermal stability)and FIG. 12 (pH stability). As a result, the enzyme was thermally stableup to about 40° C. and was stable at pHs of about 4.0 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof any of metal salts according to the assay for the enzyme activity.The results are in Table 4.

TABLE 4 Metal Relative activity Metal Relative activity ion (%) ion (%)None 100 Hg²⁺ 1 Zn²⁺ 88 Ba²⁺ 102 Mg²⁺ 98 Sr²⁺ 101 Ca²⁺ 101 Pb²⁺ 89 Co²⁺103 Fe²⁺ 96 Cu²⁺ 57 Fe³⁺ 105 Ni²⁺ 102 Mn²⁺ 106 Al³⁺ 103 EDTA 104

As evident form the results in Table 4, the enzyme activity wassignificantly inhibited by Hg²⁺ and was also inhibited by Cu²⁺. It wasalso found that the enzyme was not activated by Ca²⁺ and not inhibitedby EDTA.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:2, i.e, isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline-asparagine-glycinein the N-terminal region.

Experiment 6

Production of α-Isomaltosylglucosaccharide-Forming Enzyme from Bacillusglobisporus C11 Strain

A liquid nutrient culture medium, consisting of 4.0% (w/v) of “PINE-DEX#4”, a partial starch hydrolysate, 1.8% (w/v) of “ASAHIMEAST”, a yeastextract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumdihydrogen phosphate, dodecahydrate, 0.05% (w/v) magnesium sulfate,heptahydrate, and water was placed in 500-ml Erlenmeyer flasksrespectively in an amount of 100 ml, autoclaved at 121° C. for 20minutes to effect sterilization, cooled, inoculated with a stock cultureof Bacillus globisporus C11 strain, FERM BP-7144, and incubated at 27°C. for 48 hours under rotary shaking conditions of 230 rpm. Theresulting cultures were pooled and used as a seed culture.

About 20 L of a fresh preparation of the same nutrient culture medium asused in the above culture was placed in a 30-L fermentor, sterilized byheating, cooled to 27° C., inoculated with 1% (v/v) of the seed culture,and incubated for about 48 hours while stirring under aeration agitationconditions at 27° C. and pH 6.0 to 8.0. The resultant culture, havingabout 0.55 unit/ml of α-isomaltosylglucosaccharide-forming enzymeactivity, about 1.8 units/ml of α-isomaltosyl-transferring enzymeactivity, and about 1.1 units/ml of cyclotetrasaccharide-forming enzymeactivity, was centrifuged at 10,000 rpm for 30 min to obtain about 18 Lof a supernatant. Measurement of the supernatant revealed that it hadabout 0.51 unit/ml of α-isomaltosylglucosaccharide-forming enzymeactivity, i.e., a total enzyme activity of about 9,180 units; about 1.7units/ml of α-isomaltosyl-transferring enzyme activity, i.e., a totalenzyme activity of about 30,400 units; and about 1.1 units/ml ofcyclotetrasaccharide-forming enzyme activity, i.e., a total enzymeactivity of about 19,400 units.

Experiment 7

Preparation of Enzyme from Bacillus globisporus C11 Strain

An 18 L of the supernatant obtained in Experiment 6 was salted out withan 80% saturated ammonium sulfate solution and allowed to stand at 4° C.for 24 hours. Then the salted out sediments were collected bycentrifugation at 10,000 for 30 min, dissolved in 10 mM phosphate buffer(pH 7.5), dialyzed against a fresh preparation of the same buffer toobtain about 416 ml of a crude enzyme solution. The crude enzymesolution was revealed to have 8,440 units of theα-isomaltosylglucosaccharide-forming enzyme, about 28,000 units ofα-isomaltosyl-transferring enzyme, and about 17,700 units ofcyclotetrasaccharide-forming enzyme. When subjected to ion-exchangechromatography using “SEPABEADS FP-DA13” gel, disclosed in Experiment4-1, any of the above three types of enzymes were eluted as non-adsorbedfractions without adsorbing on the gel. The non-adsorbed fractions withthese enzymes were pooled and dialyzed against 10 mM phosphate buffer(pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove insoluble substances. The resulting supernatantwas fed to affinity chromatography using 500 ml of “SEPHACRYL HR S-200”gel to purify the enzymes. Active enzymes were adsorbed on the gel andsequentially eluted therefrom with a linear gradient decreasing from 1 Mto 0 M of ammonium sulfate and a linear gradient increasing from 0 mM to100 mM of maltotetraose, resulting in a separate elution ofα-isomaltosyl-transferring enzyme orα-isomaltosylglucosaccharide-forming enzyme, where the former enzyme waseluted with the linear gradient of ammonium sulfate at a concentrationof about 0.3 M and the latter enzyme was eluted with a linear gradientof maltotetraose at a concentration of about 30 mM. Then the fractionswith α-isomaltosyl-transferring enzyme and those withα-isomaltosylglucosaccharide-forming enzyme were separately collectedand recovered. Similarly as in the case of Bacillus globisporus C9strain in Experiment 4, it was found that nocyclotetrasaccharide-forming activity was found in any fraction in thiscolumn chromatography, and that an enzyme mixture solution of bothfractions of α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme showedcyclotetrasaccharide-forming activity, revealing that the activity offorming cyclotetrasaccharide from partial starch hydrolyzates wasexerted in collaboration with the enzyme activities of the two types ofenzymes.

Methods for separately purifying α-isomaltosylglucosaccharide-formingenzyme and α-isomaltosyl-transferring enzyme are explained below:

Experiment 7-2

Purification of α-Isomaltosylglucosaccharide-Forming Enzyme

A fraction of α-isomaltosylglucosaccharide-forming enzyme was dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate.The dialyzed solution was centrifuged to remove insoluble substances,and the resulting supernatant was fed to hydrophobic chromatographyusing 350 ml of “BUTYL-TOYOPEARL 650 M”, a gel commercialized by TosohCorporation, Tokyo, Japan. The enzyme adsorbed on the gel was elutedtherefrom at about 0.3 M ammonium sulfate with a linear gradientdecreasing from 1 M to 0 M of ammonium sulfate, followed by collectingfractions with the enzyme activity. The fractions were pooled anddialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammoniumsulfate. The resulting dialyzed solution was centrifuged to removeinsoluble substances and fed to affinity chromatography using “SEPHACRYLHR S-200” gel to purify the enzyme. The amount of enzyme activity, thespecific activity, and the yield of α-isomaltosylglucosaccharide-formingenzyme in each purification step are in Table 5.

TABLE 5 Specific activity Enzyme* activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 9,180 0.14 100Dialyzed solution after 8,440 0.60 91.9 salting out with ammoniumsulfate Eluate from ion-exchange 6,620 1.08 72.1 column chromatographyEluate from affinity 4,130 8.83 45.0 column chromatography Eluate fromhydrophobic 3,310 11.0 36.1 column chromatography Eluate from affinity2,000 13.4 21.8 column chromatography Note: The symbol “*” meansα-isomaltosylglucosaccharide-forming enzyme.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, meaning a high purity enzyme specimen.

Experiment 7-3

Purification of α-Isomaltosyl-Transferring Enzyme

A fraction of α-isomaltosyl-transferring enzyme, which had beenseparated from a fraction with α-isomaltosylglucosaccharide-formingenzyme by the affinity chromatography in Experiment 7-1, was dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate.The dialyzed solution was centrifuged to remove insoluble substances,and the resulting supernatant was fed to hydrophobic chromatographyusing 350 ml of “BUTYL-TOYOPEARL 650 M”, a gel commercialized by TosohCorporation, Tokyo, Japan. The enzyme adsorbed on the gel and was elutedtherefrom at about 0.3 M ammonium sulfate with a linear gradientdecreasing from 1 M to 0 M of ammonium sulfate, followed by collectingfractions with the enzyme activity. The fractions were pooled anddialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammoniumsulfate. The resulting dialyzed solution was centrifuged to removeinsoluble substances and fed to affinity chromatography using “SEPHACRYLHR S-200” gel to purify the enzyme. The amount of enzyme activity, thespecific activity, and the yield of the α-isomaltosyl-transferringenzyme in each purification step are in Table 6.

TABLE 6 Specific activity Enzyme* activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 30,400 0.45 100Dialyzed solution after 28,000 1.98 92.1 salting out with ammoniumsulfate Eluate from ion-exchange 21,800 3.56 71.7 column chromatographyEluate from affinity 13,700 21.9 45.1 column chromatography Eluate fromhydrophobic 10,300 23.4 33.9 column chromatography Eluate from affinity5,510 29.6 18.1 column chromatography Note: The symbol “*” meansα-isomaltosyl-transferring enzyme.Experiment 8Preparation of α-Isomaltosylglucosaccharide-Forming EnzymeExperiment 8-1Property of α-Isomaltosylglucosaccharide-Forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 7-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight in comparison with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Bio-RadLaboratories Inc., Brussels, Belgium, revealing that the enzyme had amolecular weight of about 137,000±20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 5.2±0.5.

The influence of temperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for the enzyme activity, where the influence oftemperature was conducted in the presence or absence of 1 mM Ca²⁺. Theseresults are in FIG. 13 (influence of temperature) and FIG. 14 (influenceof pH). The optimum temperature of the enzyme was about 45° C. in theabsence of Ca²⁺ and about 50° C. in the presence of 1 mM Ca²⁺ whenincubated at pH 6.0 for 60 min. The optimum pH of the enzyme was about6.0 when incubated at 35° C. for 60 min. The thermal stability of theenzyme was determined by incubating it in 20 mM acetate buffers (pH 6.0)in the presence or absence of 1 mM Ca²⁺ at prescribed temperatures for60 min, cooling the resulting enzyme solutions with water, and assayingthe remaining enzyme activity of each solution. The pH stability of theenzyme was determined by keeping it in 50 mM buffers having prescribedpHs at 4° C. for 24 hours, adjusting the pH of each solution to 6.0, andassaying the remaining enzyme activity of each solution. These resultsare respectively in FIG. 15 (thermal stability) and FIG. 16 (pHstability). As a result, the enzyme was thermally stable up to about 40°C. in the absence of Ca²⁺ and about 45° C. in the presence of 1 mM Ca²⁺.The pH stability of enzyme was in the range of about 5.0 to about 10.0.

The influence of metal ions on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in the presenceof 1 mM of any of metal salts according to the assay for the enzymeactivity. The results are in Table 7.

TABLE 7 Metal Relative activity Metal Relative activity ion (%) ion (%)None 100 Hg²⁺ 4 Zn²⁺ 91 Ba²⁺ 65 Mg²⁺ 98 Sr²⁺ 83 Ca²⁺ 109 Pb²⁺ 101 Co²⁺96 Fe²⁺ 100 Cu²⁺ 23 Fe³⁺ 102 Ni²⁺ 93 Mn²⁺ 142 Al³⁺ 100 EDTA 24

As evident form the results in Table 7, the enzyme activity was greatlyinhibited by Hg²⁺, Cu²⁺, and EDTA and was also inhibited by Ba²⁺ andSr²⁺. It was also found that the enzyme was activated by Ca²⁺ and Mn²⁺.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:1, i.e,tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucinein the N-terminal region.

The comparison of the partial amino acid sequence in the N-terminalregion with that derived from the α-isomaltosylglucosaccharide-formingenzyme from Bacillus globisporus C9 strain in Experiment 5-1 revealedthat they were the same and that the N-terminal amino acid sequence,commonly found in α-isomaltosylglucosaccharide-forming enzymes, was anamino acid sequence oftyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucineof SEQ ID NO:1 in the N-terminal region.

Experiment 8-2

Property of α-Isomaltosyl-Transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 7-3, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Bio-Rad LaboratoriesInc., Brussels, Belgium, revealing that the enzyme had a molecularweight of about 102,000±20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div. Amersham International, ArlingtonHeights, Ill., USA, and then measured for pHs of protein bands and gelto determine the isoelectric point of the enzyme, revealing that theenzyme had an isoelectric point of about 5.6±0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for the enzyme activity. These results are respectively in FIG. 17(influence of temperature) and FIG. 18 (influence of pH). The optimumtemperature of the enzyme was about 50° C. when incubated at pH 6.0 for30 min. The optimum pH of the enzyme was about 5.5 to about 6.0 whenincubated at 35° C. for 30 min. The thermal stability of the enzyme wasdetermined by incubating it in 20 mM acetate buffers (pH 6.0) atprescribed temperatures for 60 min, cooling the resulting enzymesolutions with water, and assaying the remaining enzyme activity of eachsolution. The pH stability of the enzyme was determined by keeping it in50 mM buffers having prescribed pHs at 4° C. for 24 hours, adjusting thepH of each solution to 6.0, and assaying the remaining enzyme activityof each solution. These results are respectively in FIG. 19 (thermalstability) and FIG. 20 (pH stability). As a result, the enzyme wasthermally stable up to about 40° C. and was stable at pHs of about 4.5to about 9.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof any of metal salts according to the assay for the enzyme activity.The results are in Table 8.

TABLE 8 Metal Relative activity Metal Relative activity ion (%) ion (%)None 100 Hg²⁺ 2 Zn²⁺ 83 Ba²⁺ 90 Mg²⁺ 91 Sr²⁺ 93 Ca²⁺ 91 Pb²⁺ 74 Co²⁺ 89Fe²⁺ 104 Cu²⁺ 56 Fe³⁺ 88 Ni²⁺ 89 Mn²⁺ 93 Al³⁺ 89 EDTA 98

As evident form the results in Table 8, the enzyme activity wassignificantly inhibited by Hg²⁺ and was also inhibited by Cu²⁺. It wasalso found that the enzyme was not activated by Ca²⁺ and not inhibitedby EDTA.

Amino acid analysis of the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:3, i.e., isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline-tyrosine-glycinein the N-terminal region.

The comparison of the partial amino acid sequence in the N-terminalregion with that derived from the α-isomaltosyl-transferring enzyme fromBacillus globisporus C9 strain in Experiment 5-2 revealed that they hada common amino acid sequence of isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline, as shown in SEQID NO:4 at the N-terminal region.

Experiment 9

Amino Acid Sequence of α-Isomaltosylglucosaccharide-Forming Enzyme

Experiment 9-1

Internal Amino Acid Sequence of α-Isomaltosylglucosaccharide-FormingEnzyme

A part of a purified specimen of α-isomaltosylglucosaccharide-formingenzyme, obtained by the method in Experiment 7-2, was dialyzed against10 mM Tris-HCl buffer (pH 9.0), and the dialyzed solution was dilutedwith a fresh preparation of the same buffer to give a concentration ofabout one milligram per milliliter. One milliliter of the dilute as atest sample was admixed with 10 μg of a trypsin commercialized by WakoPure Chemical Industries, Ltd., Tokyo, Japan, and incubated at 30° C.for 22 hours to hydrolyze into peptides. To isolate the peptides, theabove hydrolyzates were subjected to reverse-phase HPLC using“μ-Bondapak C18 column” with a diameter of 2.1 mm and a length of 150mm, a product of Waters Chromatography Div., MILLIPORE Corp., Milford,USA, at a flow rate of 0.9 ml/min and at ambient temperature, and usinga liner gradient of acetonitrile increasing from 8% (v/v) to 40% (v/v)in 0.1% (v/v) trifluoracetate over 120 min. The peptides eluted from thecolumn were detected by monitoring the absorbance at a wavelength of 210nm. Three peptide specimens named P64 with a retention time of about 64min, P88 with a retention time of about 88 min, and P99 with a retentiontime of about 99 min, which had been well separated from other peptides,were separately collected and dried in vacuo and then dissolved in 200μl of a solution containing 0.1% (v/v) trifluoroacetate and 50% (v/v)acetonitrile. Each peptide specimen was subjected to a protein sequencerfor analyzing amino acid sequence up to eight amino acid residues toobtain amino acid sequences of SEQ ID NOs: 5 to 7. The analyzed internalpartial amino acid sequences are in Table 9.

TABLE 9 Peptide name Internal partial amino acid sequence P64 asparticacid-alanine-serine-alanine- asparagine-valine-threonine-threonine P88tryptophane-serine-leucine-glycine- phenylalanine-methionine-asparagine-phenylalanine P99 asparagine-tyrosine-threonine-aspartic acid-alanine-tryptophane-methionine-phenylalanineExperiment 9-2Internal Amino Acid Sequence of α-Isomaltosyl-Transferring Enzyme

A part of a purified specimen of α-isomaltosyl-transferring enzyme,obtained by the method in Experiment 7-3, was dialyzed against 10 mMTris-HCl buffer (pH 9.0), and the dialyzed solution was diluted with afresh preparation of the same buffer to give a concentration of aboutone milligram per milliliter. One milliliter of the dilute as a testsample was admixed with 10 μg of “Lysyl Endopeptidase” commercialized byWako Pure Chemical Industries, Ltd., Tokyo, Japan, and allowed to reactat 30° C. for 22 hours to form peptides. The resultant mixtures weresubjected to reverse-phase HPLC to separate the peptides using“μ-Bondapak C18 column” having a diameter of 2.1 mm and a length of 150mm, a product of Waters Chromatography Div., MILLIPORE Corp., Milford,USA, at a flow rate of 0.9 ml/min and at ambient temperature, and usinga liner gradient of acetonitrile increasing from 8% (v/v) to 40% (v/v)in 0.1% (v/v) trifluoroacetate over 120 min. The peptides eluted fromthe column were detected by monitoring the absorbance at a wavelength of210 nm. Three peptide specimens named P22 with a retention time of about22 min, P63 with a retention time of about 63 min, and P71 with aretention time of about 71 min, which had been well separated from otherpeptides, were separately collected and dried in vacuo and thendissolved in 200 μl of a solution of 0.1% (v/v) trifluoroacetate and 50%(v/v) acetonitrile. Each peptide specimen was subjected to a proteinsequencer for analyzing amino acid sequence up to eight amino acidresidues to obtain amino acid sequences of SEQ ID NOs:8 to 10. Theanalyzed internal partial amino acid sequences are in Table 10.

TABLE 10 Peptide name Internal partial amino acid sequence P22glycine-asparagine-glutamic acid-methionine-arginine-asparagine-glutamine-tyrosine P63isoleucine-threonine-threonine-tryptophane- proline-isoleucine-glutamicacid-serine P71 tryptophane-alanine-phenylalanine-glycine-leucine-tryptophane-methionine-serineExperiment 10Action on Saccharides

It was tested whether the following saccharides could be used assubstrates for α-isomaltosylglucosaccharide-forming enzyme. For thepurpose, a solution of maltose, maltotriose, maltotetraose,maltopentaose, maltohexaose, maltoheptaose, isomaltose, isomaltotriose,panose, isopanose, α,α-trehalose, kojibiose, nigerose, neotrehalose,cellobiose, gentibiose, maltitol, maltotriitol, lactose, sucrose,erlose, selaginose, maltosyl glucoside, or isomaltosyl glucoside wasprepared.

To each of the above solutions was added two units/g substrate of apurified specimen of α-isomaltosylglucosaccharide-forming enzyme fromeither Bacillus globisporus C9 strain obtained by the method inExperiment 4-2, or Bacillus globisporus C11 strain obtained by themethod in Experiment 7-2, and the resulting solutions were adjusted togive a substrate concentration of 2% (w/v) and incubated at 30° C. andpH 6.0 for 24 hours. The enzyme solutions before and after the enzymaticreactions were respectively analyzed on TLC disclosed in Experiment 1 toconfirm whether the enzymes act on these substrates. The results are inTable 11.

TABLE 11 Enzymatic action Enzymatic action Enzyme of Enzyme of Enzyme ofEnzyme of Substrate C9 strain C11 strain Substrate C9 strain C11 strainMaltose + + Nigerose + + Maltotriose ++ ++ Neotrehalose + +Maltotetraose +++ +++ Cellobiose − − Maltopentaose +++ +++ Gentibiose −− Maltohexaose +++ +++ Maltitol − − Maltoheptaose +++ +++Maltotriitol + + Isomaltose − − Lactose − − Isomaltotriose − − Sucrose −− Panose − − Erlose + + Isopanose ++ ++ Selaginose − − α,α-Trehalose − −Maltosylglucoside ++ ++ Kojibiose + + Isomaltosylglucoside − − Note:Before and after the enzymatic reaction, the symbols “−”, “+”, “++”, and“+++” mean that it showed no change, it showed a slight reduction of thecolor of substrate spot and the formation of other reaction product, itshowed a high reduction of the color of substrate spot and the formationof other reaction product, and it showed a substantial disappearance ofthe color of substrate spot and the formation of other reaction product,respectively.

As evident from Table 11, it was revealed that theα-isomaltosylglucosaccharide-forming enzyme well acted on saccharideshaving a glucose polymerization degree of at least three and having amaltose structure at their non-reducing ends, among the saccharidestested. It was also found that the enzyme slightly acted on saccharides,having a glucose polymerization degree of two, such as maltose,kojibiose, nigerose, neotrehalose, maltotriitol, and erlose.

Experiment 11

Reaction Product from Maltooligosaccharide

Experiment 11-1

Preparation of Reaction Product

To an aqueous solution containing one percent (w/v) of maltose,maltotriose, maltotetraose, or maltopentaose as a substrate was added apurified specimen of α-isomaltosylglucosaccharide-forming enzymeobtained by the method in Experiment 7-2 in an amount of two units/gsolid for maltose and maltotriose, 0.2 unit/g solid for maltotetraosefor maltotetraose, and 0.1 unit/g solid for maltopentaose, followed bythe incubation at 35° C. and pH 6.0 for eight hours. After a 10-minincubation at 100° C., the enzymatic reaction was suspended. Theresulting reaction solutions were respectively measured for saccharidecomposition on HPLC using “YMC-PACK ODS-AQ303”, a column commercializedby YMC Co., Ltd., Tokyo, Japan, at a column temperature of 40° C. and aflow rate of 0.5 ml/min of water, and using as a detector “RI-8012”, adifferential refractometer commercialized by Tosoh Corporation, Tokyo,Japan. The results are in Table 12.

TABLE 12 Saccharide as reaction Substrate product Maltose MaltotrioseMaltotetraose Maltopentaose Glucose 8.5 0.1 0.0 0.0 Maltose 78.0 17.90.3 0.0 Maltotriose 0.8 45.3 22.7 1.9 Maltotetraose 0.0 1.8 35.1 19.2Maltopentaose 0.0 0.0 3.5 34.4 Maltohexaose 0.0 0.0 0.0 4.6 Isomaltose0.5 0.0 0.0 0.0 Gluco- 8.2 1.2 0.0 0.0 sylmaltose Gluco- 2.4 31.5 6.80.0 sylmaltotriose X 0.0 2.1 30.0 11.4 Y 0.0 0.0 1.4 26.8 Z 0.0 0.0 0.01.7 Others 0.6 0.1 0.2 0.0 Note: In the table, glucosylmaltose meansα-isomaltosylglucose alias 6²-O-α-glucosylmaltose or panose;glucosylmaltotriose means α-isomaltosylglucose alias6³-O-α-glucosylmaltotriose; X means the α-isomaltosylglucotriose inExperiment 11-2, alias 6⁴-O-α-glucosylmaltotetraose; Y means theα-isomaltosylglucotetraose in Experiment 11-2, alias6⁵-O-α-glucosylmaltopentaose; and Z means an unidentified saccharide.

As evident from the results in Table 12, it was revealed that, after theaction of the α-isomaltosylglucosaccharide-forming enzyme, glucose andα-isomaltosylglucose alias 6²-O-α-glucosylmaltose or panose were mainlyformed from maltose as a substrate; and from maltotriose as a substrate,maltose and α-isomaltosylglucose alias 6³-O-α-glucosylmaltotriose weremainly formed along with small amounts of glucose, maltotetraose,α-isomaltosylglucose alias 6²-O-α-glucosylmaltose or panose, and theproduct X. Also, it was revealed that maltotriose and the product X weremainly formed from maltotetraose as a substrate along with small amountsof maltose, maltopentaose, α-isomaltosylglucose alias6³-O-α-glucosylmaltotriose, and the product Y; and that maltotetraoseand the product Y were mainly formed from maltopentaose as a substratealong with small amounts of maltotriose, maltohexaose, and the productsX and Z.

The product X as a main product from maltotetraose as a substrate andthe product Y as a main product from maltopentaose as a substrate wererespectively isolated and purified as follows: The products X and Y wererespectively purified on HPLC using “YMC PACK ODS-A R355-15S-15 12A”, aseparatory HPLC column commercialized by YMC Co., Ltd., Tokyo, Japan, toisolate a specimen of the product X having a purity of at least 99.9%from the reaction product of maltotetraose in a yield of about 8.3%,d.s.b., and a specimen of the product Y having a purity of at least99.9% from the reaction product of maltopentaose in a yield of about11.5%, d.s.b.

Experiment 11-2

Structural Analysis on Reaction Product

Using the products X and Y obtained by the method in Experiment 11-1,they were subjected to methyl analysis and NMR analysis in a usualmanner. The results on their methyl analyses are in Table 13. For theresults on their NMR analyses, FIG. 21 is a ¹H-NMR spectrum for theproduct X and FIG. 22 is for the product Y. The ¹³C-NMR spectra for theproducts X and Y are respectively FIGS. 23 and 24. The assignment of theproducts X and Y are tabulated in Table 14.

TABLE 13 Analyzed Ratio methyl compound Product X Product Y2,3,4-Trimethyl compound 1.00 1.00 2,3,6-Trimethyl compound 3.05 3.982,3,4,6-Tetramethyl compound 0.82 0.85

Based on these results, the product X, formed from maltotetraose via theaction of the α-isomaltosylglucosaccharide-forming enzyme, was revealedas a pentasaccharide, in which a glucose residue bounds via theα-linkage to OH-6 of glucose at the non-reducing end of maltotetraose,i.e., α-isomaltosylmaltotriose alias 6⁴-O-α-glucosylmaltotetraose,represented by Formula 1.α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-D-Glcp  Formula1:

The product Y formed from maltopentaose was revealed as ahexasaccharide, in which a glucosyl residue bounds via the α-linkage toOH-6 of glucose at the non-reducing end of maltopentaose, i.e.,α-isomaltosylmaltotetraose alias 6⁵-O-α-glucosylmaltopentaose,represented by Formula 2.α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-D-Glcp  Formula2:

TABLE 14 Glucose Carbon Chemical shift on NMR (ppm) number numberProduct X Product Y a 1a 100.8 100.8 2a 74.2 74.2 3a 75.8 75.7 4a 72.272.2 5a 74.5 74.5 6a 63.2 63.1 b 1b 102.6 102.6 2b 74.2 74.2 3b 75.875.7 4b 72.1 72.1 5b 74.0 74.0 6b 68.6 68.6 c 1c 102.3 102.3 2c 74.274.2 3c 76.0 76.0 4c 79.6 79.5 5c 73.9 73.9 6c 63.2 63.1 d 1d 102.2102.3 2d 74.0(α), 74.4(β) 74.2 3d 76.0 76.0 4d 79.8 79.5 5d 73.9 73.9 6d63.2 63.1 e 1e 94.6(α), 98.5(β) 102.1 2e 74.2(α), 76.7(β) 74.0(α),74.4(β) 3e 75.9(α), 78.9(β) 76.0 4e 79.6(α), 79.4(β) 79.8 5e 72.6(α),77.2(β) 73.9 6e 63.4(α), 63.4(β) 63.1 f 1f 94.6(α), 98.5(β) 2f 74.2(α),76.7(β) 3f 76.0(α), 78.9(β) 4f 79.6(α), 79.5(β) 5f 72.6(α), 77.2(β) 6f63.3(α), 63.3(β)

Based on these results, it was concluded that theα-isomaltosylglucosaccharide-forming enzyme acts onmaltooligosaccharides as shown below:

-   -   (1) The enzyme acts on, as a substrate, a maltooligosaccharide        having a glucose polymerization degree of at least two, where        glucose residues are linked via the α-1,4 linkage; and catalyzes        the intermolecular 6-glucosyl-transferring reaction in such a        manner of transferring a glucosyl residue at the non-reducing        end of a maltooligosaccharide molecule to C-6 of the glucosyl        residue at the non-reducing end of another maltooligosaccharide        molecule to form both an α-isomaltosylglucosaccharide alias        6-O-α-glucosylmaltooligosaccharide, having a 6-O-α-glucosyl        residue and a higher glucose polymerization degree by one as        compared with the intact substrate, and a maltooligosaccharide        with a lower glucose polymerization degree by one as compared        with the intact substrate; and    -   (2) The enzyme slightly catalyzes the 4-glucosyl-transferring        reaction and forms both a maltooligosaccharide, having a higher        glucose polymerization degree by one as compared with the intact        substrate, and a maltooligosaccharide having a lower glucose        polymerization degree by one as compared with the intact        substrate.        Experiment 12        Test on the Formation of Reducing Power

The following test was carried out to examine whetherα-isomaltosylglucosaccharide-formation enzyme had an ability of formingreducing power. To a 1% (w/v) aqueous solution of maltotetraose as asubstrate was added 0.25 unit/g substrate, d.s.b., of either of purifiedspecimens of α-isomaltosylglucosaccharide-forming enzyme from Bacillusglobisporus C9 strain obtained by the method in Experiment 4-2 andBacillus globisporus C11 strain obtained by the method in Experiment7-2, and incubated at 35° C. and pH 6.0. During the enzymatic reaction,a portion of each reaction solution was sampled at prescribed timeintervals and measured for reducing powder after keeping the sampledsolutions at 100° C. for 10 min to suspend the enzymatic reaction.Before and after the enzymatic reaction, the reducing saccharide contentand the total sugar content were respectively quantified by theSomogyi-Nelson's method and the anthrone-sulfuric acid reaction method.The percentage of forming reducing power was calculated by the followingequation:

Equation: $\begin{matrix}{{Percentage}\mspace{14mu}{of}\mspace{14mu}{forming}} \\{{reducing}\mspace{14mu}{power}\mspace{11mu}(\%)}\end{matrix} = {( {\frac{AR}{AT} - \frac{BR}{BT}} ) \times 100}$AR:  Reducing  sugar  content  after  enzymatic  reaction.AT:  Total  sugar  content  after  enzymatic  reaction.BR:  Reducing  sugar  content  before  enzymatic  reaction.BT:  Total  sugar  content  before  enzymatic  reaction.

The results are in Table 15.

TABLE 15 Percentage of forming Reaction reducing power (%) time Enzymeof Enzyme of (hour) C9 strain C11 strain 0 0.0 0.0 1 0.0 0.1 2 0.1 0.0 40.1 0.1 8 0.0 0.0

As evident from the results in Table 15, it was revealed thatα-isomaltosylglucosaccharide-forming enzyme does not substantiallyincrease the reducing power of the reaction product when allowed to acton maltotetraose as a substrate; the enzyme does not exhibit hydrolyzingactivity or only has an undetectable level of such activity.

Experiment 13

Test on the Formation of Dextran

To study whether α-isomaltosylglucosaccharide-forming enzyme has theability of forming dextran, it was tested in accordance with the methodin Bioscience Biotechnology and Biochemistry, Vol. 56, pp. 169–173(1992). To a 1% (w/v) aqueous solution of maltotetraose as a substratewas added 0.25 unit/g substrate, d.s.b., of either of purified specimensof α-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporusC9 strain obtained by the method in Experiment 4-2 or Bacillusglobisporus C11 strain obtained by the method in Experiment 7-2 andincubated at 35° C. and pH 6.0 for four or eight hours. After completionof the enzymatic reaction, the reaction was suspended by heating at 100°C. for 15 min. Fifty microliters of each of the reaction mixtures wereplaced in a centrifugation tube and then admixed and sufficientlystirred with 3-fold volumes of ethanol, followed by standing at 4° C.for 30 min. Thereafter, each of the resulting mixtures was centrifugedat 15,000 rpm for five minutes and, after removing the supernatant, theresulting sediment was admixed with one milliliter of 75% (v/v) ethanolsolution and stirred for washing. The resulting each solution wascentrifuged to remove supernatant, dried in vacuo, and then admixed andsufficiently stirred with one milliliter of deionized water. The totalsugar content, in terms of glucose, of each resulting solution wasquantified by the phenol-sulfuric acid method. As a control, the totalsugar content was determined similarly as in the above except for usingeither of purified specimens of α-isomaltosylglucosaccharide-formingenzyme from Bacillus globisporus C9 strain and Bacillus globisporus C11strain, which had been inactivated at 100° C. for 10 min. The content ofdextran formed was calculated by the following equation:Content of dextran formed (mg/ml)=[(Total sugar content for testsample)−(Total sugar content for control sample)]×20  Equation:

The results are in Table 16.

TABLE 16 Reaction Content of dextran formed (mg/ml) time Enzyme ofEnzyme of (hour) C9 strain C11 strain 4 0.0 0.0 8 0.0 0.0

As evident from the results in Table 16, it was revealed that theα-isomaltosylglucosaccharide-forming enzyme does not substantially havethe action of forming dextran or only has an undetectable level of suchactivity because it did not form dextran when allowed to act onmaltotetraose.

Experiment 14

Specificity of Transfer Acceptor

A variety of saccharides were tested whether they could be used astransferring-acceptors for the α-isomaltosylglucosaccharide-formingenzyme. A solution of D-glucose, D-xylose, L-xylose, D-galactose,D-fructose, D-mannose, D-arabinose, D-fucose, L-sorbose, L-rhamnose,methyl-α-glucopyranoside, methyl-β-glucopyranoside,N-acetyl-glucosamine, sorbitol, α,α-trehalose, isomaltose,isomaltotriose, cellobiose, gentibiose, maltitol, lactose, sucrose,α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin was prepared.

To each solution with a saccharide concentration of 1.6% was added“PINE-DEX #100”, a partial starch hydrolysate, as a saccharide donor, togive a concentration of 4%, and admixed with one unit/g saccharidedonor, d.s.b., of either of purified specimens ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9strain obtained by the method in Experiment 4-2 and Bacillus globisporusC11 strain obtained by the method in Experiment 7-2, and incubated at30° C. and pH 6.0 for 24 hours. The reaction mixtures after theenzymatic reactions were analyzed on gas chromatography (abbreviated as“GLC” hereinafter) for monosaccharides and disaccharides as acceptors,and on HPLC for trisaccharides and higher saccharides as acceptors toconfirm whether these saccharides could be used as their transferacceptors. In the case of performing GLC, the following apparatuses andconditions were used: GLC apparatus, “GC-16A” commercialized by ShimadzuCorporation, Tokyo, Japan; column, a stainless-steel column, 3 mm indiameter and 2 m in length, packed with 2% “SILICONE OV-17/CHROMOSOLVW”, commercialized by GL Sciences Inc., Tokyo, Japan; carrier gas,nitrogen gas at a flow rate of 40 ml/min under temperature conditions ofincreasing from 160° C. to 320° C. at an increasing temperature rate of7.5° C./min; and detection, a hydrogen flame ionization detector. In thecase of HPLC analysis, the apparatuses and conditions used were: HPLCapparatus, “CCPD” commercialized by Tosoh Corporation, Tokyo, Japan;column, “ODS-AQ-303” commercialized by YMC Co., Ltd., Tokyo, Japan;eluent, water at a flow rate of 0.5 ml/min; and detection, adifferential refractometer. The results are in Table 17.

TABLE 17 Product of Product of saccharide saccharide transferringtransferring reaction reaction Enzyme Enzyme Enzyme Enzyme of C9 of C11of C9 of C11 Saccharide strain strain Saccharide strain strainD-Glucose + + Sorbitol − − D-Xylose ++ ++ α,α-Trehalose ++ ++ L-Xylose++ ++ Isomaltose ++ ++ D-Galactose + + Isomaltotriose ++ ++D-Fructose + + Cellobiose ++ ++ D-Mannose − − Gentibiose ++ ++D-Arabinose ± ± Maltitol ++ ++ D-Fucose + + Lactose ++ ++ L-Sorbose + +Sucrose ++ ++ L-Rhamnose − − α-Cyclodextrin − − Methyl-α- ++ ++β-Cyclodextrin − − glucopyranoside Methyl-β- ++ ++ γ-Cyclodextrin − −glucopyranoside N-Acetyl- + + glucosamine Note: In the table, thesymbols “−”, “±”, “+”, and “++” mean that no saccharide-transferredproduct was detected through transferring reaction to acceptor; asaccharide-transferred product was detected in an amount of less thanone percent through transfer reaction to acceptor; asaccharide-transferred product was detected in an amount of at least onepercent but less than ten percent through transferring reaction toacceptor; and a saccharide-transferred product was detected in an amountof at least ten percent through transferring reaction to acceptor.

As evident from the results in Table 17, it was revealed that theα-isomaltosylglucosaccharide utilizes different types of saccharides astransfer acceptors, particularly, the enzyme most preferably transfers asaccharide to D-/L-xylose, methyl-α-glucopyranoside,methyl-β-glucopyranoside, α,α-trehalose, isomaltose, isomaltotriose,cellobiose, gentibiose, maltitol, lactose, and sucrose; next toD-glucose, D-fructose, D-fucose, L-sorbose, and N-acetylglucosamine; andthen to D-arabinose.

The properties of the α-isomaltosylglucosaccharide-forming enzymedescribed above were compared with those of a previously reported enzymehaving 6-glucosyl-transferring action; a dextrin dextranase disclosed in“Bioscience Biotechnology and Biochemistry”, Vol. 56, pp. 169–173(1992); and a transglucosidase disclosed in “Nippon Nogeikagaku Kaishi”,Vol. 37, pp. 668–672 (1963). The results are in Table 18.

TABLE 18 α-Isomaltosyl- glucosaccharide- Dextrin forming enzymedextranase Transglucosidase Property C9 strain C11 strain (Control)(Control) Hydrolysing Negative Negative Negative Significantly abilitypositive Ability of Negative Negative Positive Negative forming dextranOptimum pH 6.0–6.5 6.0 4.0–4.2 3.5 Inhibition Positive Positive NegativeNegative by EDTA

As evident from Table 18, the α-isomaltosylglucosaccharide-formingenzyme had outstandingly novel physicochemical properties completelydifferent from those of known dextrin dextranase and transglucosidase.

Experiment 15

Formation of Cyclotetrasaccharide

Using different saccharides, the formation of cyclotetrasaccharide byα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme was tested: It was prepared a solutionof maltose, maltotriose, maltotetraose, maltopentaose, amylose, solublestarch, “PINE-DEX #100” (a partial starch hydrolyzate commercialized byMatsutani Chemical Ind., Tokyo, Japan), or glycogen from oystercommercialized by Wako Pure Chemical Industries Ltd., Tokyo, Japan. Toeach of these solutions with a respective concentration of 0.5% wereadded one unit/g solid of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporusC11 strain obtained by the method in Experiment 7-2, and 10 units/gsolid of a purified specimen of α-isomaltosyl-transferring enzyme fromBacillus globisporus C11 strain obtained by the method in Experiment7-3, and the resulting mixtures were subjected to an enzymatic reactionat 30° C. and pH 6.0. The enzymatic conditions were the following foursystems:

-   -   (1) After 24-hour incubation of the        α-isomaltosylglucosaccharide-forming enzyme with any of the        saccharide solutions, the enzyme was inactivated by heating, and        then the α-isomaltosyl-transferring enzyme was allowed to act on        any of the resulting mixtures for 24 hours and inactivated by        heating;    -   (2) After 24-hour simultaneous incubation of the        α-isomaltosylglucosaccharide-forming enzyme and the        α-isomaltosyl-transferring enzyme with any of the saccharide        solutions, then the enzymes were inactivated by heating;    -   (3) After 24-hour incubation of the        α-isomaltosylglucosaccharide-forming enzyme alone with any of        the saccharide solutions, then the enzyme was inactivated by        heating; and    -   (4) After 24-hour incubation of the α-isomaltosyl-transferring        enzyme alone with any of the saccharide solutions, then the        enzyme was inactivated by heating.

To determine the formation level of cyclotetrasaccharide in eachreaction mixture after the heating, the reaction mixtures were subjectedto a similar treatment with α-glucosidase and glucoamylase as inExperiment 1 to hydrolyze the remaining reducing oligosaccharides,followed by the quantitation of cyclotetrasaccharide on HPLC. Theresults are in Table 19.

TABLE 19 Yield of cyclotetrasaccharide (%) Substrate A B C D Maltose 4.04.2 0.0 0.0 Maltotriose 10.2 12.4 0.0 0.0 Maltotetraose 11.3 21.5 0.00.0 Maltopentaose 10.5 37.8 0.0 0.0 Amylose 3.5 31.6 0.0 0.0 Solublestarch 5.1 38.2 0.0 0.0 Partial starch 6.8 63.7 0.0 0.0 hydrolyzateGlycogen 10.2 86.9 0.0 0.0 Note: The symbols “A”, “B”, “C” and “D” meanthat α-isomaltosylglucosaccharide-forming enzyme was first allowed toact on a substrate and then α-isomaltosyl-transferring enzyme wasallowed acted on the resulting mixture, theα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme were allowed to coact on a substrate,only α-isomaltosylglucosaccharide-forming enzyme was allowed to act on asubstrate, and only α-isomaltosyl-transferring enzyme was allowed to acton a substrate.

As evident from the results in Table 19, no cyclotetrasaccharide wasformed from any of the saccharides tested by the action of either of theα-isomaltosylglucosaccharide-forming enzyme or theα-isomaltosyl-transferring enzyme, but cyclotetrasaccharide was formedby the coaction of these enzymes. It was revealed that the formationlevel was relatively low as about 11% or lower when theα-isomaltosyl-transferring enzyme was allowed to act on the saccharidesafter the action of α-isomaltosylglucosaccharide-forming enzyme, whilethe level increased when the enzymes were simultaneously allowed to acton any of the saccharides tested, particularly, it increased to about87% and about 64% when allowed to act on glycogen and partial starchhydrolyzate, respectively.

Based on the reaction properties of theα-isomaltosylglucosaccharide-forming enzyme and theα-isomaltosyl-transferring enzyme, the formation mechanism ofcyclotetrasaccharide by the coaction of the above enzymes is estimatedas follows:

-   -   (1) The α-isomaltosylglucosaccharide-forming enzyme acts on a        glucose residue at the non-reducing end of an α-1,4 glucan chain        of glycogen, partial starch hydrolyzates, etc., and        intermolecularly transfers the glucose residue to OH-6 of a        glucose residue at the non-reducing end of another intact α-1,4        glucan chain of glycogen, partial starch hydrolyzates, etc., to        form an α-1,4 glucan chain having an α-isomaltosyl residue at        the non-reducing end;    -   (2) The α-isomaltosyl-transferring enzyme acts on the        α-1,4-glucan chain having an α-isomaltosyl residue at the        non-reducing end and intermolecularly transfers the isomaltosyl        residue to C-3 of glucose residue at the non-reducing end of        another intact α-1,4 glucan chain having an isomaltosyl residue        at the non-reducing end to form an α-1,4 glucan chain having an        isomaltosyl-1,3-isomaltosyl residue at the non-reducing end;    -   (3) Then, the α-isomaltosyl-transferring enzyme acts on the        α-1,4 glucan chain having an isomaltosyl-1,3-isomaltosyl residue        at the non-reducing end and releases the        isomaltosyl-1,3-isomaltosyl residue from the α-1,4 glucan chain        via the intramolecular transferring reaction to cyclize the        released isomaltosyl-1,3-isomaltosyl residue into        cyclotetrasaccharide;    -   (4) From the remaining released α-1,4 glucan chain,        cyclotetrasaccharide is formed through the sequential steps (1)        to (3), resulting in an estimation that the coaction of        α-isomaltosylglucosaccharide-forming enzyme and        α-isomaltosyl-transferring enzyme in such a cyclic manner as        indicated above increases the yield of cyclotetrasaccharide.        Experiment 16        Influence of Liquefaction Degree of Starch

A 15% corn starch suspension was prepared, admixed with 0.1% calciumcarbonate, adjusted to pH 6.0, and then mixed with 0.2 to 2.0% per gramstarch of “TERMAMYL 60L”, an α-amylase commercialized by Novo IndutriA/S, Copenhagen, Denmark, followed by an enzymatic reaction at 95° C.for 10 min. Thereafter, the reaction mixture was autoclaved at 120° C.for 20 min, promptly cooled to about 35° C. to obtain a liquefied starchwith a DE (dextrose equivalent) of 3.2 to 20.5. To the liquefied starchwere added two units/g solid of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporusC11 strain obtained by the method in Experiment 7-2, and 20 units/gsolid of a purified specimen of α-isomaltosyl-transferring enzyme fromBacillus globisporus C11 strain obtained by the method in Experiment7-3, followed by the incubation at 35° C. for 24 hours. After completionof the reaction, the reaction mixture was heated at 100° C. for 15 minto inactivate the remaining enzymes. Then, the reaction mixture thusobtained was treated with α-glucosidase and glucoamylase similarly as inExperiment 1 to hydrolyze the remaining reducing oligosaccharides,followed by quantifying the formed cyclotetrasaccharide on HPLC. Theresults are in Table 20.

TABLE 20 Amount of α-amylase Yield of per starch (%) DEcyclotetrasaccharide (%) 0.2 3.2 54.5 0.4 4.8 50.5 0.6 7.8 44.1 1.0 12.539.8 1.5 17.3 34.4 2.0 20.5 30.8

As evident from the results in Table 20, it was revealed that theformation of cyclotetrasaccharide by the coaction ofα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme is influenced by the liquefactiondegree of starch, i.e., the lower the liquefaction degree or the lowerthe DE, the higher the yield of cyclotetrasaccharide from starchbecomes. On the contrary, the higher the liquefaction degree or thehigher the DE, the lower the yield of cyclotetrasaccharide from starchbecomes. It was revealed that a suitable liquefaction degree is a DE ofabout 20 or lower, preferably, a DE of about 12 or lower, morepreferably, a DE of about 5 or lower.

Experiment 17

Influence of Concentration of Partial Starch Hydrolyzate

Aqueous solutions of “PINE-DEX #100”, a partial starch hydrolyzate witha DE of about 2 to about 5, having a final concentration of 0.5 to 40%,were prepared and respectively admixed with one unit/g solid of apurified specimen of α-isomaltosylglucosaccharide-forming enzyme fromBacillus globisporus C11 strain obtained by the method in Experiment7-2, and 10 units/g solid of a purified specimen ofα-isomaltosyl-transferring enzyme from Bacillus globisporus C11 strainobtained by the method in Experiment 7-3, followed by the coaction ofthese enzymes at 30° C. and pH 6.0 for 48 hours. After completion of thereaction, each reaction mixture was heated at 100° C. for 15 min toinactivate the remaining enzymes, and then treated with α-glucosidaseand glucoamylase similarly as in Experiment 1 to hydrolyze the remainingreducing oligosaccharides, followed by quantifying the formedcyclotetrasaccharide on HPLC. The results are in Table 18.

TABLE 18 Concentration of Yield of PINE-DEX (%) cyclotetrasaccharide (%)0.5 63.6 2.5 62.0 5 60.4 10 57.3 15 54.6 20 51.3 30 45.9 40 39.5

As evident from the results in Table 21, the yield ofcyclotetrasaccharide was about 64% at a low concentration of 0.5%, whileit was about 40% at a high concentration of 40%. The fact showed thatthe yield of cyclotetrasaccharide increases depending on theconcentration of partial starch hydrolyzate as a substrate. The resultrevealed that the yield of cyclotetrasaccharide increases as theconcentration of partial starch hydrolyzate decreases.

Experiment 18

Influence of the Addition of Cyclodextrin Glucanotransferase

Fifteen percent of aqueous solutions of “PINE-DEX #100”, a partialstarch hydrolyzate, were prepared and admixed with one unit/g solid of apurified specimen of α-isomaltosylglucosaccharide-forming enzyme fromBacillus globisporus C11 strain obtained by the method in Experiment7-2, 10 units/g solid of a purified specimen ofα-isomaltosyl-transferring enzyme from Bacillus globisporus C11 strainobtained by the method in Experiment 7-3, and 0–0.5 unit/g solid of acyclodextrin glucanotransferase (CGTase) from a microorganism of thespecies Bacillus stearothermophilus, followed by the coaction of theseenzymes at 30° C. and pH 6.0 for 48 hours. After completion of thereaction, the reaction mixture was heated at 100° C. for 15 min toinactivate the remaining enzymes, and then treated with α-glucosidaseand glucoamylase similarly as in Experiment 1 to hydrolyze the remainingreducing oligosaccharides, followed by quantifying the formedcyclotetrasaccharide on HPLC. The results are in Table 22.

TABLE 22 Amount of CGTase added Yield of (unit) cyclotetrasaccharide (%)0 54.6 2.5 60.1 5 63.1 10 65.2

As evident from the Table 22, it was revealed that the addition ofCGTase increases the yield of cyclotetrasaccharide.

Experiment 19

Preparation of Cyclotetrasaccharide

About 100 L of a 4% (w/v) aqueous solution of a corn phytoglycogencommercialized by Q.P. Corporation, Tokyo, Japan, was prepared, adjustedto pH 6.0 and 30° C., and then admixed with one unit/g solid of apurified specimen of α-isomaltosylglucosaccharide-forming enzyme fromBacillus globisporus C11 strain obtained by the method in Experiment7-2, 10 units/g solid of a purified specimen ofα-isomaltosyl-transferring enzyme from Bacillus globisporus C11 strainobtained by the method in Experiment 7-3, followed by the incubation for48 hours. After completion of the reaction, the reaction mixture washeated at 100° C. for 10 min to inactivate the remaining enzymes, and aportion of the reaction mixture was sampled and then quantified on HPLCto calculate the yield of cyclotetrasaccharide, revealing that itcontained 84% cyclotetrasaccharide with respect to saccharides, d.s.b.The reaction mixture was adjusted to pH 5.0 and 45° C., and then treatedwith α-glucosidase and glucoamylase similarly as in Experiment 1 tohydrolyze the remaining reducing oligosaccharides, etc. The resultingmixture was adjusted to pH 5.8 by the addition of sodium hydroxide andthen incubated at 90° C. for one hour to inactivate the remainingenzymes and filtered to remove insoluble substances. The filtrate wasconcentrated using a reverse osmosis membrane to give a concentration ofabout 16%, d.s.b., and the concentrate was in a usual manner decolored,desalted, filtered, and concentrated to obtain about 6.2 kg of asaccharide solution with a solid content of about 3,700 g.

The saccharide solution was fed to a column packed with about 225 L of“AMBERLITE CR-1310 (Na-form)”, an ion-exchange resin commercialized byJapan Organo Co., Ltd., Tokyo, Japan, and chromatographed at a columntemperature of 60° C. and a flow rate of about 45 L/h. While thesaccharide composition of the eluate from the column was being monitoredby HPLC as described in Experiment 1, fractions of cyclotetrasaccharidewith a purity of at least 98% were collected, and in a usual mannerdesalted, decolored, filtered, and concentrated to obtain about 7.5 kgof a saccharide solution with a solid content of about 2,500 g solids.HPLC measurement for saccharide composition of the saccharide solutionrevealed that it contained cyclotetrasaccharide with a purity of about99.5%.

Experiment 20

Crystallization of Cyclotetrasaccharide in Aqueous Solution

A fraction of cyclotetrasaccharide with a purity of at least 98%,obtained by the method in Experiment 19, was concentrated by evaporationto give a concentration of about 50%, d.s.b. About five kilograms of theconcentrate was placed in a cylindrical plastic vessel and thencrystallized to obtain a white crystalline powder by lowering thetemperature of the concentrate from 65° C. to 20° C. over about 20 hoursunder gentle rotatory conditions. FIG. 25 is a microscopic photograph ofsuch cyclotetrasaccharide. The above crystallized concentrate wasseparated by a centrifugal filter to obtain 1,360 g of a crystallineproduct by wet weight, which was then further dried at 60° C. for threehours to obtain 1,170 g of a crystalline powder of cyclotetrasaccharide.HPLC measurement of the crystalline powder revealed that it containedcyclotetrasaccharide with a quite high purity of 99.9% or over.

When analyzed on powder x-ray diffraction analysis, thecyclotetrasaccharide in a crystalline powder form had a diffractionspectrum having characteristic main diffraction angles (2θ) of 10.1°,15.2°, 20.3°, and 25.5° in FIG. 26. The Karl Fischer method of thecrystalline powder revealed that it had a moisture content of 13.0%,resulting in a finding that it was a crystal of cyclotetrasaccharidehaving five or six moles of water per one mole of cyclotetrasaccharide.

The thermogravimetric analysis of the cyclotetrasaccharide in acrystalline form gave a thermogravimetric curve in FIG. 27. Based on therelationship between the weight change and the temperature, it wassuccessively found that the weight reduction corresponding to four orfive moles of water was observed up to a temperature of 150° C., theweight reduction corresponding to one mole of water at around 250° C.,and the weight reduction corresponding to the decomposition ofcyclotetrasaccharide at a temperature of about 280° C. or higher. Theseresults confirmed that the crystalline cyclotetrasaccharide, penta- orhexa-hydrate, of the present invention releases four or five moles ofwater to change into its crystalline monohydrate form when heated up to150° C. at normal pressure, and further releases one mole of water tochange into its anhydrous crystal form until reaching 250° C.

Experiment 21

Conversion into Crystalline Cyclotetrasaccharide, Monohydrate

Crystalline cyclotetrasaccharide, penta- or hexa-hydrate, in a powderform, obtained by the method in Experiment 20, was placed in a glassvessel, and kept in an oil bath, which had been preheated to 140° C.,for 30 min. Being quite different from the result from the powder x-raydiffraction analysis of intact cyclotetrasaccharide, penta- orhexa-hydrate, free from heat treatment, the powder x-ray analysis of thecyclotetrasaccharide powder thus obtained gave a characteristicdiffraction spectrum having main diffraction angles (2θ) of 8.3°, 16.6°,17.0°, and 18.2° in FIG. 28. The Karl Fischer method of the crystallinepowder revealed that it had a moisture content of about 2.7%, revealingthat it was a crystal of cyclotetrasaccharide having one mole of waterper one mole of cyclotetrasaccharide. The thermogravimetric analysis ofthe cyclotetrasaccharide in a crystalline powder form gave athermogravimetric curve in FIG. 29. Based on the relationship betweenthe weight change and the temperature, it was found that the weightreduction corresponding to one mole of water was observed at atemperature of about 270° C. and further observed the weight reductioncorresponding to the decomposition of cyclotetrasaccharide per se at atemperature of about 290° C. or higher. These results confirmed that thecyclotetrasaccharide crystal in this experiment was crystallinecyclotetrasaccharide, monohydrate.

Experiment 22

Conversion into Anhydrous Crystal

Crystalline cyclotetrasaccharide, penta- or hexa-hydrate, in a powderform, obtained by the method in Experiment 20, was dried in vacuo at 40°C. or 120° C. for 16 hours. The Karl Fischer method of the resultingcrystalline powders revealed that the one dried at 40° C. had a moisturecontent of about 4.2%, while the other dried at 120° C. had a moisturecontent of about 0.2%, meaning that it was substantially anhydrous.Being quite different from the results from the powder x-ray diffractionanalyses of the crystalline cyclotetrasaccharide, penta- orhexa-hydrate, and the crystalline cyclotetrasaccharide, monohydrate,before drying in vacuo, the powder x-ray analysis of the abovecyclotetrasaccharide, dried in vacuo at 40° C. and 120° C., gavecharacteristic diffraction spectra having main diffraction angles (2θ)of 10.8°, 14.7°, 15.0°, 15.7°, and 21.5° in FIG. 30 for 40° C. and FIG.31 for 120° C. Although there found difference in peak levels betweenthe two diffraction spectra, they had substantially the same peakdiffraction angles and they were crystallographically judged to besubstantially the same crystalline monohydrate. The fact that the baselines of the diffraction spectra exhibited a mountain-like pattern andthe crystallinity of the crystalline monohydrate was lower than those ofcrystalline cyclotetrasaccharide, penta- or hexa-hydrate, andcrystalline cyclotetrasaccharide, monohydrate, before drying in vacuo.This revealed the existence of an amorphous cyclotetrasaccharide. Basedon this, the cyclotetrasaccharide powder with a moisture content ofabout 4.2%, obtained by drying in vacuo at 40° C., was estimated to be amixture powder of an amorphous cyclotetrasaccharide with such a moisturecontent and anhydrous crystalline cyclotetrasaccharide. These datarevealed that crystalline cyclotetrasaccharide, penta- or hexa-hydrate,was converted into those in an anhydrous amorphous- and an anhydrouscrystalline-forms when dried in vacuo. The thermogravimetric analysis ofanhydrous crystalline cyclotetrasaccharide with a moisture content of0.2%, which was conducted similarly as in Experiment 20, observed onlythe weight reduction as shown in FIG. 32, deemed to be induced by theheat decomposition at a temperature of about 270° C. or higher.

Experiment 23

Saturation Concentration of Cyclotetrasaccharide in Water

To study the saturation concentration of cyclotetrasaccharide in waterat 10–90° C., 10 ml of water was placed in a glass vessel with a sealcap, and then mixed with cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Experiment 20, in an excessive amount over alevel of complete dissolution at respective temperatures, cap-sealed,and stirred for two days while keeping at respective temperatures of10–90° C. until reaching saturation. The resulting each saturatedsolution of cyclotetrasaccharide was membrane filtered to removeundissolved cyclotetrasaccharide, and each filtrate was then examinedfor moisture content by the drying loss method to determine a saturationconcentration of cyclotetrasaccharide at respective temperatures. Theresults are in Table 23.

TABLE 23 Temperature (° C.) Saturation concentration (%) 10 30.3 30 34.250 42.6 70 53.0 90 70.5Experiment 24Thermostability

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 20, was dissolved in water into a 10% (w/v)aqueous cyclotetrasaccharide solution, and eight milliliter aliquots ofwhich were placed in glass test tubes, followed by sealing and thenheating the test tubes at 120° C. for 30 to 90 min. After the heating,the aqueous solutions were cooled under atmospheric conditions andmeasured for coloration degree and determined for purity on HPLC. Thecoloration degree of each solution was evaluated based on the absorbancein a cell with a 1-cm light pass at a wavelength of 480 nm. The resultsare in Table 24.

TABLE 24 Heating time Coloration degree Purity (min) (A_(480 nm)) (%) 00.00 100 30 0.00 100 60 0.00 100 90 0.00 100

As evident from the results in Table 24, it was revealed thatcyclotetrasaccharide is a thermostable saccharide because an aqueouscyclotetrasaccharide solution was not colored, and the purity of thesaccharide composition in the solution was not lowered even when heatedat a high temperature of 120° C.

Experiment 25

pH Stability

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 20, was dissolved in 20 mM buffers withdifferent pHs into 4% (w/v) cyclotetrasaccharide solutions with pHs of 2to 10. Eight milliliters of each solution was placed in a glass testtube, followed by sealing and then heating the test tube at 100° C. for24 hours. After the heating, each solution was cooled and measured forcoloration degree and determined for purity on HPLC. The colorationdegree was evaluated based on the absorbance in a cell with a 1-cm lightpass at a wavelength of 480 nm. The results are in Table 25.

TABLE 25 pH Coloration degree Purity (type of buffer) (A_(480 nm)) (%) 2.0 (Acetate buffer) 0.00 93  3.0 (Acetate buffer) 0.00 100  4.0(Acetate buffer) 0.00 100  5.0 (Acetate buffer) 0.00 100  6.0 (Tris-HClbuffer) 0.00 100  7.0 (Tris-HCl buffer) 0.00 100  8.0 (Tris-HCl buffer)0.00 100  9.0 (Ammonium buffer) 0.00 100 10.0 (Ammonium buffer) 0.00 100

As evident from the results in Table 25, a cyclotetrasaccharide aqueoussolution was not colored even when heated at 100° C. for 24 hours in awide pH range from 2 to 10, and the purity of the saccharide compositionin each solution was not lowered at all in a pH range from 3 to 10, eventhough the purity was slightly lowered at pH 2. These facts revealedthat cyclotetrasaccharide is highly stable in a relatively wide pHrange, i.e., an acid pH range from 3 to 5, a neutral pH range from 6 to8, and an alkaline pH range from 9 to 10.

Experiment 26

Amino Carbonyl Reaction

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 20, was dissolved in water, and then admixedwith a commercialized special grade glycine and phosphate buffer, andthe resulting mixture was then adjusted to pH 7.0 with 50 mM phosphatebuffer to obtain a 10% (w/v) cyclotetrasaccharide solution containing 1%(w/v) glycine. Four milliliter aliquots of the resulting solution wereplaced in glass test tubes, sealed, and heated at 120° C. for 30 to 90min. After allowing to stand for cooling at ambient temperature, each ofthe resulting solutions was measured for coloration degree to examinetheir amino carbonyl reactivity. The coloration degree was evaluatedbased on the absorbance in a cell with 1-cm light pass at a wavelengthof 480 nm. The results are in Table 26.

TABLE 26 Heating time (min) Coloration degree (A_(480 nm)) 0 0.00 300.00 60 0.00 90 0.00

As evident from the results in Table 26, cyclotetrasaccharide was notcolored even when heated in the presence of glycine, meaning that thesaccharide does not induce browning with glycine, i.e.,cyclotetrasaccharide is a stable saccharide which does not induce theamino carbonyl reaction, also known as the Maillard reaction.

Experiment 27

Amino Carbonyl Reaction

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 20, and a polypeptone commercialized byNihonseiyaku K.K., Tokyo, Japan, were dissolved in deionized water toobtain a 10% (w/v) cyclotetrasaccharide solution containing 5% (w/v)polypeptone. Four milliliter aliquots of the resulting solution wereplaced in glass test tubes, sealed, and heated at 100° C. for 30 to 90min. After allowing to stand for cooling at ambient temperature, each ofthe resulting solution was measured for coloration degree to examinetheir amino carbonyl reactivity. In parallel, as a control, a solutionwith only polypeptone was provided and treated similarly as above. Thecoloration degree was evaluated based on the level of the absorbance,which had been measured in a cell with 1-cm light pass at a wavelengthof 480 nm, minused that of the control. The results are in Table 27.

TABLE 27 Heating time (min) Coloration degree (A_(480 nm)) 0 0.00 300.00 60 0.00 90 0.00

As evident from the results in Table 27, it was revealed thatcyclotetrasaccharide did not induce browning with polypeptone whenheated in the presence of polypeptone, i.e., the saccharide is a stablesaccharide which substantially does not induce the amino carbonylreaction.

Experiment 28

Inclusion Action

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 20, was dissolved in deionized water into a 20%(w/v) aqueous cyclotetrasaccharide solution. To 100 g aliquots of theaqueous solution was added 2 g of methanol, 3 g of ethanol, or 4.6 gacetic acid to be included by the cyclotetrasaccharide. Thereafter, eachof the resulting solutions was filtered to remove non-inclusionproducts, and the filtrates were dried in vacuo. As a control, similarinclusion products were prepared by using “ISOELITE™ P”, a branchedcyclodextrin commercialized by Maruha K.K., Tokyo, Japan, which wereknown to have inclusion ability.

To measure the amount of the inclusion products in the resultinglyophilized powders, one gram of each of the powders was dissolved infive milliliters of water and extracted after admixing with fivemilliliters of diethylether. The extraction was repeated, and theresulting extracts were quantified on gas chromatography. The resultsare in Table 28.

TABLE 28 Inclusion Inclusion amount (mg/g lyophilized powder) productCyclotetrasaccharide ISOELITE P (control) Methanol 6.71 2.92 Ethanol17.26 8.92 Acetic acid 67.74 30.57

As evident from the results in Table 28, it was revealed thatcyclotetrasaccharide has an inclusion ability of about 2-folds higherthan that of the branched cyclodextrin by weight.

Experiment 29

Sweetening Power

A crystalline cyclotetrasaccharide, penta- or hexa-hydrate, obtained bythe method in Experiment 20, was dissolved in deionized water into a 10%(w/v) aqueous cyclotetrasaccharide solution for a standard solution.Varying the concentration of sucrose, e.g., a commercialized granulatedsugar, a sensory test was done with five panelists. As a result, thesweetening power of cyclotetrasaccharide was about 20% of that ofsucrose.

Experiment 30

Digestion Test

Using a crystalline cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Experiment 20, the digestibility ofcyclotetrasaccharide in vitro by salivary amylase, synthetic gastricjuice, amylopsin, or intestinal mucosal enzyme was tested in accordancewith the method as reported by K. Okada et al. in JOURNAL OF JAPANESESOCIETY OF NUTRITION AND FOOD SCIENCE, Vol. 43, No. 1, pp. 23–29 (1990).As a control, maltitol known as a substantially non-digestive saccharidewas used. The results are in Table 29.

TABLE 29 Decomposition percentage (%) by digestive enzyme MaltitolDigestive enzyme Cyclotetrasaccharide (Control) Salivary amylase 0.0 0.0Synthetic 0.0 0.0 gastric juice Amylopsin 0.0 0.0 Small intestinal 0.744.0 mucosal enzyme

As evident from the results in Table 29, cyclotetrasaccharide was notcompletely digested by salivary amylase, synthetic gastric juice, andamylopsin, but slightly digested by intestinal mucosal enzyme in adigestibility level as low as 0.74% that corresponded to ⅕ of that ofmaltitol as a control. These results confirmed that cyclotetrasaccharideis a quite undigestible saccharide.

Experiment 31

Fermentation Test

Using a crystalline cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Experiment 20, the fermentability ofcyclotetrasaccharide using an internal content of rat cecum was testedin accordance with the method by T. Oku in “Journal of NutritionalScience and Vitaminology”, Vol. 37, pp. 529–544 (1991). The internalcontent of rat cecum was collected by anesthetizing a Wister male ratwith ether, anatomizing the rat, collecting the internal content underanaerobic conditions, and suspending the resultant with 4-fold volumesof a 0.1 M aqueous solution of sodium bicarbonate. Thecyclotetrasaccharide was added to the internal content of rat cecum inan amount of about 7% by weight, and the levels of cyclotetrasaccharideremained just after and 12 hours after the addition of the internalcontent were quantified on gas chromatography. As a result, the levelsof cyclotetrasaccharide of the former and the latter were respectively68.0 mg and 63.0 mg per one gram of the internal content of rat cecum,meaning that 93% of cyclotetrasaccharide remained intact. These dataconfirmed that cyclotetrasaccharide is a substantially non-fermentablesaccharide.

Experiment 32

Assimilation Test

Using a crystalline cyclotetrasaccharide, penta- or hexa-hydrate,obtained by the method in Experiment 20, the assimilability ofcyclotetrasaccharide by an internal content of rat cecum was studied inaccordance with the method disclosed in “Intestinal Flora and DietaryFactors”, edited by Tomotari MITSUOKA, published by Japan ScientificSocieties Press, Tokyo, Japan, (1984). About 10⁷ CFU (colony formingunits) of pre-cultured fresh microorganisms were inoculated into fivemilliliters of PYF medium with 0.5% cyclotetrasaccharide, and culturedat 37° C. for four days under anaerobic conditions. As a control,glucose was used as an easily assimilable saccharide. The assimilabilitywas judged as negative (−) when the post culture had a pH of 6.0 orhigher, and judged as positive (+) when the post culture had a pH ofbelow 6.0. The judgement of assimilability was confirmed by measuringthe content of saccharide, which remained in the culture, on theanthrone method to determine the reduction level of saccharide. Theresults are in Table 30.

TABLE 30 Strain of intestinal Assimilability microorganismCyclotetrasaccharide Glucose (control) Bacteroides vulgatus − + JCM 5826Bifidobacterium adolescentis − + JCM 1275 Clostridium perfringens − +JCM 3816 Escherichia coli − + IFO 3301 Eubacterium aerofaciens − + ATCC25986 Lactobacillus acidophilus − + JCM 1132

As evident from the results in Table 30, it was confirmed thatcyclotetrasaccharide was not assimilated by any of the strains tested,but glucose as a control was assimilated by all the strains tested. Thedata confirmed that cyclotetrasaccharide is a saccharide which is notsubstantially assimilated by intestinal microorganisms.

Experiment 33

Acute Toxicity Test

The acute toxicity of a crystalline cyclotetrasaccharide, penta- orhexa-hydrate, obtained by the method in Experiment 20, was tested byorally administering to mice. As a result, it was revealed thatcyclotetrasaccharide had relatively low toxicity and did not cause thedeath of mice even when administered at the highest possible dose. Basedon this, the LD₅₀ of cyclotetrasaccharide was at least 50 g/kg mousebody weight, though the value was so accurate.

Based on the results in Experiments 30 to 33, cyclotetrasaccharide isnot substantially assimilated or absorbed by living bodies when orallytaken, and it can be expected to be used as a non- or low-caloric ediblematerial in diet sweeteners; fillers for sweeteners with a relativelyhigh sweetening power; viscosity agents, fillers, and bodies for dietfood products; and further it can be used as an edible fiber and a foodmaterial for substituting fats.

Experiment 34

Comparative Experiment on the Dehydration Degree of Moisture andPulverization of Dehydrated Product by Non-Reducing Saccharide

The non-reducing saccharides used in this experiment were anhydrouscrystalline cyclotetrasaccharide; crystalline cyclotetrasaccharide,monohydrate; anhydrous amorphous cyclotetrasaccharide; crystallinecyclotetrasaccharide, penta- or hexa-hydrate; anhydrous crystallineα,α-trehaose, anhydrous amorphous α,α-trehaose; and crystallineα,α-trehaose, dihydrate. The crystalline cyclotetrasaccharide, penta- orhexa-hydrate, was prepared by the method in Experiment 20. The anhydrouscrystalline cyclotetrasaccharide; crystalline cyclotetrasaccharide,monohydrate; and anhydrous amorphous cyclotetrasaccharide wererespectively prepared by the methods in Examples A-1, A-2, and A-3. Asthe crystalline α,α-trehalose, dihydrate, a commercially available“TREHA®” commercialized by Hayashibara Shoji Inc., Okayama, Japan, wasused. The anhydrous crystalline α,α-trehalose and the anhydrousamorphous α,α-trehalose used in this experiment were prepared from acommercialized crystalline α,α-trehalose, dihydrate, using the methodsdisclosed in Examples for Reference 1 and 3 in Japanese Patent Kokai No.170,221/94.

To four parts by weight of a plain yogurt with a moisture content ofabout 77% was added either of the above saccharides in an amount of 11to 16 parts by weight. The resultant mixtures were respectively placedin vats, allowed to stand at 25° C. for 24 hours, and macroscopicallyobserved their change on standing. The judgement was conducted in such amanner of sufficiently dehydrating the resulting mixtures to besolidified, subjected to a pulverizer for pulverization, and evaluatedas “◯”, when the solids were easily pulverized; “Δ”, when thedehydration of the resulting mixtures was rather insufficient and thepulverization of solids was substantially difficult, though theresulting mixtures were solidified; and “x”, when the dehydration of theresulting mixtures was insufficient and could not be pulverized by apulverizer. The results are in Table 31.

TABLE 31 Weight (part by weight) of saccharide Water to four parts byweight of plain yogurt content with a moisture content of about 77%Saccharide (before use) 11 12 13 14 15 16 A 0.2 X Δ

B 2.7 X X X Δ

C 0.3 X Δ

D 13.0 X X X X X X E 0.3 X X X X Δ

F 0.8 X X X X Δ

G 9.7 X X X X X X Note: The symbol “A” means anhydrous crystallinecyclotetrasaccharide; B, crystalline cyclotetrasaccharide, monohydrate;C, anhydrous amorphous cyclotetrasaccharide; D, crystallinecyclotetrasaccharide, penta- or hexa-hydrate; E, anhydrous crystallineα,α-trehalose; F, anhydrous amorphous α,α-trehalose; and G, crystallineα,α-trehalose, dihydrate.

As evident from the results in Table 31, it was revealed that anhydrouscrystalline cyclotetrasaccharide; crystalline cyclotetrasaccharide,monohydrate; and anhydrous amorphous cyclotetrasaccharide solidified theplain yogurt in a lesser amount than those required in anhydrouscrystalline α,α-trehalose and anhydrous amorphous α,α-trehalose; andfacilitated the pulverization of the solidified yogurts. The resultingpowders had satisfactory properties. Based on these, anhydrouscrystalline cyclotetrasaccharide; crystalline cyclotetrasadcharide,monohydrate; and anhydrous amorphous cyclotetrasaccharide, which are thecyclotetrasaccharides with dehydrating ability, are suitably used asdehydrating agents, particularly, anhydrous crystallinecyclotetrasaccharide and anhydrous amorphous cyclotetrasaccharide have asuperior dehydrating ability.

Experiment 35

Dehydrating Action by Cyclotetrasaccharide with Dehydrating Ability

Anhydrous crystalline cyclotetrasaccharide; crystallinecyclotetrasaccharide, monohydrate; anhydrous amorphouscyclotetrasaccharide; and crystalline cyclotetrasaccharide, penta- orhexa-hydrate were experimented in detail on their dehydrating actions,particularly, moisture absorption abilities against saccharides andchanges on standing. As a control, anhydrous crystalline α,α-trehalose,anhydrous amorphous α,α-trehalose, and crystalline α,α-trehalose,dihydrate, were used as saccharides. The experiments were as follows:Cyclotetrasaccharide and α,α-trehalose preparations, prepared by themethod in Experiment 34, were respectively sieved into a powder with agranular size of about 100–150 μm. One gram of each of the resultingpowders was placed in a plastic petri dish, 5 cm in diameter, placed ina desiccator controlled at a relative humidity of 60% or 75%, andallowed to stand at 25° C. for a week while sampling the saccharides ata prescribed time interval for quantifying the moisture content (%) bythe Karl Fisher method. The results are in Table 32.

TABLE 32 Relative humidity (%) Days after treatment Saccharide whentreated 0 1 2 3 7 A 60 0.2 10.2 10.3 10.3 10.4 75 0.2 13.9 14.4 14.414.3 B 60 2.7  2.7  2.8  2.8  2.9 75 2.7 14.0 14.0 14.1 14.1 C 60 0.313.7 13.8 13.9 14.1 75 0.3 14.2 13.6 13.7 13.7 D 60 13.0  13.1 13.1 13.113.2 75 13.0  13.1 13.1 13.1 13.1 E 60 — — — — — 75 0.3  9.7  9.6  9.8 9.8 F 60 — — — — — 75 0.8  9.8  9.7  9.8  9.7 G 60 — — — — — 75 9.7 9.7  9.7  9.8  9.8 Note: The symbol “A” means anhydrous crystallinecyclotetrasaccharide; B, crystalline cyclotetrasaccharide, monohydrate;C, anhydrous amorphous cyclotetrasaccharide; D, crystallinecyclotetrasaccharide, penta- or hexa-hydrate; E, anhydrous crystallineα,α-trehalose; F, anhydrous amorphous α,α-trehalose; and G, crystallineα,α-trehalose, dihydrate. The symbol “—” means “not tested”.

As evident from the results in Table 32, both crystallinecyclotetrasaccharide, monohydrate; and crystalline cyclotetrasaccharide,penta- or hexa-hydrate, did not substantially absorb moisture even after1-week standing at a relatively humidity of 60%, while anhydrouscrystalline cyclotetrasaccharide and anhydrous amorphouscyclotetrasaccharide reached almost their saturated moisture absorptionlevels on day 1. The moisture absorption level of anhydrous crystallinecyclotetrasaccharide is about 10% of its weight, while that of anhydrousamorphous cyclotetrasaccharide is about 14% of its weight. Powder X-raydiffraction analysis of each saccharide after 1-week standing revealedthat they showed the same predominant diffraction angles as those ofthem before standing tests and did not change in their crystallineforms. It was revealed that the saccharides absorbed moisture at arelative humidity of 60% or lower, but they did not contain water as acrystal water.

When allowed to stand at a relative humidity of 75%, anhydrouscrystalline cyclotetrasaccharide, crystalline cyclotetrasaccharide,monohydrate, and anhydrous amorphous cyclotetrasaccharide reached almosttheir saturated moisture absorption levels after 1-day standing,similarly as in anhydrous crystalline α,α-trehalose and anhydrousamorphous α,α-trehalose. In this case, the moisture absorption levels ofthese cyclotetrasaccharides were about 14% of each of their weights,while that of α,α-trehalose was not higher than 10% of its weight,revealing that the former is superior to the latter. All the saccharidestested kept their powder forms and did not become sticky or flowing.Powder X-ray diffraction analysis of anhydrous crystallinecyclotetrasaccharide, crystalline cyclotetrasaccharide, monohydrate; andanhydrous amorphous cyclotetrasaccharide, penta- or hepta-hydrate, after1-week standing revealed that these saccharides showed a differentpredominant diffraction pattern from those of them before standingtests, which corresponded to the diffraction pattern of crystallinecyclotetrasaccharide, penta- or hexa-hydrate. Based on the results, itwas revealed that anhydrous cyclotetrasaccharides are converted intocrystalline cyclotetrasaccharide, penta- or hexa-hydrate, afterincorporating water as a crystal water at a relative humidity of atleast 75%.

Thus, it was concluded that the cyclotetrasaccharide with an effectivedehydrating ability according to the present invention can beadvantageously used as a strong dehydrating agent for food products,pharmaceuticals, cosmetics, and their materials and processingintermediates.

Experiment 36

Comparison of the Effect of Anhydrous Crystalline Cyclotetrasaccharideand Crystalline Cyclotetrasaccharide, Penta- or Hexa-Hydrate, onBacterial Contamination of Gelatinized Starch

Four parts by weight of a rice flour were dissolved in six parts byweight of water, and the mixture was poured into a container surroundedwith woods whose inner surface was covered with a wet cloth, and steamedat 105° C. for 10 min to obtain a gelatinized starch. To the resultinggelatinized starch was added six parts by weight of either crystallinecyclotetrasaccharide, penta- or hexa-hydrate, prepared by the method inExperiment 20, or anhydrous crystalline cyclotetrasaccharide prepared bythe method in Example A-1. The mixture was mixed with a mixer andfurther mixed to homogeneity with two parts by weight of a starchhydrolyzate, shaped, and roughly dried for two hours, while blowing 40°C. hot air to the contents, to obtain “gyuhi” (a rice paste with sugar).

After allowed to stand at ambient temperature of 25° C. under openconditions, there were found colonies of Aspergillus niger in gyuhiprepared with crystalline cyclotetrasaccharide, penta- or hexa-hydrate,at 15 days on standing, but found no bacterial contamination in gyuhiwith anhydrous crystalline cyclotetrasaccharide even at 30 days onstanding.

Gyuhi, prepared with anhydrous cyclotetrasaccharide, at 30 days onstanding was cut and macroscopically observed its cross section,revealing that the surface of the product slightly solidified and hadcrystallized cyclotetrasaccharide but the internal texture kept itssemi-transparency, adequate gloss and viscosity similarly as in theproduct just after processed. Upon X-ray diffraction pattern of thecrystal on the surface of the product revealed that anhydrouscrystalline cyclotetrasaccharide was converted into crystallinecyclotetrasaccharide, penta- or hexa-hydrate.

Based on the results, it was concluded that the cyclotetrasaccharidewith dehydrating ability acts as a dehydrating agent for, preventsbacterial contamination of, and inhibits the retrogradation ofgelatinized starch. These characteristics can be advantageously used inproducts with gelatinized starch such as a gyuhi or flour paste.

The following Examples A explain the cyclotetrasaccharide withdehydrating ability used in the present invention, and Examples Bexplain the uses of the saccharide in detail:

EXAMPLE A-1

Process for Producing Anhydrous Crystalline Cyclotetrasaccharide

A microorganism of the species Bacillus globisporus C11 strain, FERMBP-7144, was cultured by a fermentor for 48 hours in accordance with themethod in Experiment 6. After completion of the culture, the resultingculture was filtered with an SF membrane to remove cells and to collectabout 18 L of a culture supernatant. Then the culture supernatant wasconcentrated with a UF membrane to collect about one liter of aconcentrated enzyme solution containing 9.0 units/ml ofα-isomaltosylglucosaccharide-forming enzyme and 30.2 units/ml ofα-isomaltosyl-transferring enzyme. A tapioca starch was prepared into anabout 25% starch suspension which was then admixed with 0.2% per gramstarch, d.s.b., of “NEO-SPITASE”, an α-amylase commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan, and enzymatically reacted at 85° C. to90° C. for about 20 min. Thereafter, the reaction mixture was autoclavedat 120° C. for 20 min and then promptly cooled to about 35° C. to obtaina liquefied solution with a DE of about four. To the liquefied solutionwas added 0.25 ml per gram starch, d.s.b., of the above concentratedenzyme solution, containing α-isomaltosylglucosaccharide-forming enzymeand α-isomaltosyl-transferring enzyme, and further added 10 units/gstarch, d.s.b., of a CGTase commercialized by Hayashibara BiochemicalLaboratories, Inc., Okayama, Japan, followed by the enzymatic reactionat pH 6.0 and 35° C. for 48 hours. The reaction mixture was heated toand kept at 95° C. for 30 min and then adjusted to pH 5.0 and 50° C. andadmixed with 300 units/g starch, d.s.b., of “TRANSGLUCOSIDASE L AMANO™”,an α-glucosidase commercialized by Amano Pharmaceutical Co., Ltd.,Aichi, Japan, followed by an enzymatic reaction for 24 hours. Further,the reaction mixture was mixed with 30 units/g starch, d.s.b.,“GLUCOZYME”, a glucoamylase commercialized by Nagase Biochemicals, Ltd.,Kyoto, Japan, and then enzymatically reacted for 17 hours. The reactionmixture thus obtained was heated to and kept at 95° C. for 30 min, andthen cooled and filtered to obtain a filtrate. The resulting filtratewas in a conventional manner decolored with an activated charcoal,desalted and purified with ion exchangers in H- and OH-forms, and thenconcentrated to obtain a 60% cyclotetrasaccharide syrup in a yield ofabout 90% to the material starch, d.s.b. According to Experiment 19, thesyrup containing cyclotetrasaccharide was subjected to a column packedwith 225 L of “AMBERLITE CR-1310 (Na-form)”, a strong-acidcation-exchange resin commercialized by Japan Organo Co., Ltd., Tokyo,Japan, and chromatographed at a flow rate of about 45 L/min whilekeeping the inner column temperature at 60° C. While the saccharidecomposition of the eluate was monitoring on HPLC described in Experiment1, fractions rich in cyclotetrasaccharide were collected, pooled, andpurified to obtain a high cyclotetrasaccharide content solution in ayield of about 21% to the material starch, d.s.b. The solution containedabout 98% cyclotetrasaccharide, d.s.b. After concentrated into an about90% solution, the resulting concentrate was placed in a crystallizer,admixed with two percent of anhydrous crystalline cyclotetrasaccharideas a seed, and dried in vacuo while keeping at 120° C. for 16 hours toobtain anhydrous crystalline cyclotetrasaccharide with a moisturecontent of about 0.2%. Since the product has a strong dehydratingability, it can be advantageously used in dehydrating methods for foodproducts, chemical products, pharmaceuticals, and their materials andprocessing intermediates.

EXAMPLE A-2

Process for Producing Crystalline Cyclotetrasaccharide, Monohydrate

A potato starch was prepared into an about 20% starch suspension,admixed with calcium carbonate to give a final concentration of 0.1%,adjusted to pH 6.5, further admixed with 0.3% per gram starch, d.s.b.,of “TERMAMYL 60L”, an α-amylase commercialized by Novo Industri A/S,Copenhagen, Denmark, and then enzymatically reacted at 95° C. for about15 min. Thereafter, the mixture was autoclaved at 120° C. for 20 min andthen promptly cooled to about 35° C. to obtain a liquefied solution witha DE of about four. To the liquefied solution were added 0.25 ml pergram starch, d.s.b., of a concentrated enzyme solution containingα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme, and 10 units/g starch, d.s.b., of aCGTase commercialized by Hayashibara Biochemical Laboratories Inc.,Okayama, Japan, followed by the enzymatic reaction at pH 6.0 and 35° C.for 48 hours. The reaction mixture was heated to and kept at 95° C. for30 min and then adjusted to pH 5.0 and 50° C., followed by the enzymaticreaction for 24 hours after the addition of 300 units/g solid of“TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase commercialized by AmanoPharmaceutical Co., Ltd., Aichi, Japan, and then the enzymatic reactionfor 17 hours after the addition of 30 units/g solid of “GLUCOZYME”, aglucoamylase commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan.The resulting reaction mixture was heated to and kept at 95° C. for 30min, and then cooled and filtered. The filtrate thus obtained was in aconventional manner decolored with an activated charcoal, desalted, andpurified with ion exchangers in H- and OH-forms, and then concentratedto obtain a 60% cyclotetrasaccharide syrup in a yield of about 90% tothe material starch, d.s.b. According to the method in Example A-1, thesyrup was chromatographed, followed by collecting fractions with apurity of cyclotetrasaccharide of at least 98%. Then, according to themethod in Experiment 20, the fractions were pooled and concentrated byan evaporator into a concentrate having a solid concentration of about50%. Five kilograms of the concentrate was placed in a cylindricalplastic container and cooled from 65° C. to 20° C. over about 20 hoursunder gentle stirring conditions to effect crystallization, followed byobtaining a powdery crystalline cyclotetrasaccharide, penta- orhexa-hydrate. The powder was placed in a glass container which was thenkept in an oil bath, preheated to 140° C., for 30 min. The dried productwas pulverized by a pulverizer to obtain a powdery crystallinecyclotetrasaccharide, monohydrate, with a moisture content of about 7%.Since the product has a strong dehydrating ability, it can beadvantageously used in dehydrating methods for food products, chemicalproducts, pharmaceuticals, and their materials and processingintermediates.

EXAMPLE A-3

Process for Producing Anhydrous Amorphous Cyclotetrasaccharide

Fractions containing cyclotetrasaccharide with a purity of at least 98%,obtained according to the method in Example A-1, were in a usual mannerdesalted, decolored, and filtered to obtain a concentrate having a solidconcentration of 50%. The concentrate thus obtained was promptly freezedat −80° C., lyophilized, and further dried in vacuo at 80° C. for threehours. The resulting dried product was pulverized by a pulverizer toobtain a powdery anhydrous amorphous cyclotetrasaccharide with amoisture content of about 0.3%. FIG. 33 is an X-ray diffraction patternof the powder. Since the product has a strong dehydrating ability, itcan be advantageously used in dehydrating methods for food products,chemical products, pharmaceuticals, and their materials and processingintermediates.

EXAMPLE A-4

Process for Producing Anhydrous Crystalline Tetrasaccharide from Panose

About 100 L of an aqueous solution of panose, produced from starch andcommercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan, was adjusted to give a concentration of 4% (w/v), pH 6.0, and to30° C., admixed with two units/g panose, d.s.b., of a purifiedα-isomaltosyl-transferring enzyme obtained by the method in Experiment7, and enzymatically reacted for 48 hours. Thereafter, the reactionmixture was heated at 100° C. for 10 min to inactivate the remainingenzyme and sampled for analyzing the percentage of cyclotetrasaccharidein the saccharide composition on HPLC, and revealed to be about 44%. Thereaction mixture after the heat treatment was adjusted to pH 5.0 and 45°C. and subjected to an enzymatic reaction for 24 hours after theaddition of 1,500 units/g solid of “TRANSGLUCOSIDASE L AMANO™”, anα-glucosidase commercialized by Amano Pharmaceutical Co., Ltd., Aichi,Japan, and 75 units/g solid of “GLUCOZYME”, a glucoamylasecommercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, to hydrolyzethe remaining reducing oligosaccharides, etc. Thereafter, the resultingmixture was adjusted to pH 5.8 with sodium hydroxide, incubated at 90°C. for one hour to inactivate the remaining enzymes, and filtered toremove insoluble substances. The filtrate was concentrated to give asolid concentration of about 16% using a reverse osmotic membrane, andthe concentrate was in a usual manner decolored, desalted, filtered, andconcentrated to obtain about 6.1 kg of a saccharide solution with asolid content of about 3,650 g. The saccharide solution waschromatographed according to the method in Example A-1, followed bycollecting fractions with a purity of cyclotetrasaccharide of at least98%. The fractions were pooled and in a usual manner decolored,desalted, filtered, and concentrated to obtain about 3 kg of asaccharide solution with a solid content of about 1,000 g. HPLC analysisfor saccharide composition of the saccharide solution revealed that thesolution contained cyclotetrasaccharide with a purity of about 99.2%.The cyclotetrasaccharide solution thus obtained was concentrated by anevaporator into a concentrate having a solid concentration of about 50%.About two kilograms of the concentrate was placed in a cylindricalplastic container, and cooled from 65° C. to 20° C. over about 20 hoursunder gently rotatory conditions to effect crystallization, followed bydrying the formed crystal to obtain crystalline cyclotetrasaccharide,penta- or hexa-hydrate. The crystal thus obtained was further dried invacuo at 120° C. for 16 hours into anhydrous crystallinecyclotetrasaccharide with a moisture content of about 0.2%. Since theproduct has a strong dehydrating ability, it can be advantageously usedin dehydrating methods for food products, chemical products,pharmaceuticals, and their materials and processing intermediates.

EXAMPLE B-1

Dehydrating Agent

Fifteen grams aliquots of a powdery anhydrous crystallinecyclotetrasaccharide, obtained by the method in Example A-1, wererespectively injected into a moisture permeable small paper bag toobtain a dehydrating agent. The product is advantageously used as anagent for dehydrating the inner atmosphere of moisture-proof containerswhich house seasoned sea layers, cookies, etc., and also can bearbitrarily used in combination with a deoxidizer(s) in dried or oilyfood products to stably store them.

EXAMPLE B-2

Sugar with Dehydrating Agent

To 50 parts by weight of sugar was added one part by weight of a hydrouscrystalline cyclotetrasaccharide powder obtained by the method inExample A-4, followed by mixing with a high-speed rotary mixer. Onekilogram aliquots of the resulting mixture were respectively placed in apolyethylene bag, followed by deaerating the gas space in the bag, heatsealing the opening of the bag to obtain a sugar composition with adehydrating agent. In the product, the dehydrating agent absorbsmoisture on the surface of microcrystalline sugars, and this is creditedwith preventing them from adhering and solidifying, and with ensuringits stable shelf-life. The product can be used as a seasoning inpreparing cooked/processed foods.

EXAMPLE B-3

Salt with Dehydrating Agent

To 100 parts by weight of salt was added one part by weight of a hydrouscrystalline cyclotetrasaccharide powder obtained by the method inExample A-1, followed by mixing on a high-speed rotary mixer. Onekilogram aliquots of the resulting mixture were respectively placed in apolyethylene bag, followed by deaerating the gas spaces of the bags andheat sealing their openings to obtain a salt composition with adehydrating agent. In the product, the dehydrating agent absorbsmoisture on the surface of microcrystalline salts, and this is creditedwith preventing them from adhering and solidifying and with ensuring itsstable shelf-life. The product can be used as a seasoning in preparingcooked/processed foods.

EXAMPLE B-4

“Soboro-Gyuhi” (a Rice Paste Like Soboro “a Dried Fish Meat Flake”)

Four parts by weight of a rice flour were dissolved in six parts byweight of water, poured into a container surrounded with woods whoseinner surface was covered with a wet cloth, steamed at 100° C. for 20min, kneaded with one part by weight of sugar and six parts by weight ofan anhydrous crystalline cyclotetrasaccharide powder obtained by themethod in Example A-1, and sufficiently mixed with two parts by weightof a hydrolyzed starch syrup. The mixture was shaped, allowed to standat room temperature for 16 hours to convert the saccharide intocrystalline cyclotetrasaccharide, penta- or hexa-hydrate, lightly rolledto make cracks on the surface of the resultant product to obtain thecaptioned product. The product has a satisfactory flavor and taste, issubstantially free of bacterial contamination, and keeps its highquality for a relatively long period of time.

EXAMPLE B-5

Confectionery of Sweet Potato

A sweet potato was sliced into pieces of about 1-cm in thickness,steamed, cooled, and sprinkled with an anhydrous crystallinecyclotetrasaccharide powder obtained by the method in Example A-3 toconvert the saccharide into crystalline cyclotetrasaccharide, penta- orhexa-hydrate. Thus, a confectionery of sweet potato, which the convertedsaccharide adhered unto the surface, was produced, and it had asatisfactory stability, flavor, and taste.

EXAMPLE B-6

Powdered Cream

One part by weight of a fresh cream and three parts by weight of ananhydrous crystalline cyclotetrasaccharide powder, obtained by themethod in Example A-1, were mixed and transferred to a vat, and allowedto stand for two days to form a block while converting the saccharideinto crystalline cyclotetrasaccharide, penta- or hexa-hydrate. The blockwas pulverized by a cutter and classified to obtain a powdered creamwith a satisfactory flavor and taste. Thus, the product can be used tosweeten coffee and tea and used as a material for processing premixes,frozen desserts, cakes, and candies, as well as a therapeutic nutritionfor intubation feedings.

EXAMPLE B-7

Powdered Brandy

Two parts by weight of a brandy were mixed with 10 parts by weight ofpullulan and seven parts by weight of an anhydrous crystallinecyclotetrasaccharide powder obtained by the method in Example A-1. Theresulting mixture was allowed to stand for two days to form a blockwhile converting the saccharide into crystalline cyclotetrasaccharide,penta- or hexa-hydrate. The block was subjected to a cutter forpulverization and classified to obtain a powdered brandy with asatisfactory flavor and taste, i.e., a powdered flavor with an adequatesweetness and a sufficient flavor of brandy when tasted in your mouth.The product can be used for imparting flavor to tea and advantageouslyused as a material for confectioneries such as premixes and candies.Also the product can be shaped by a granulator or a tabletting machineinto a granule or tablet for use.

EXAMPLE B-8

Powdered Miso

Two parts by weight of akamiso were mixed with four parts by weight of apowdery crystalline cyclotetrasaccharide, monohydrate, obtained by themethod in Example A-1. The mixture was poured over a metal plate with aplural semispheric dimples, allowed to stand at ambient temperatureovernight to solidify the contents. The solids were removed from thedimples to obtain solid misos, about four grams each, which were thensubjected to a pulverizer to obtain the captioned product. The productcan be arbitrarily used as a seasoning for instant soups and also usedas a solid seasoning and a miso confectionery.

EXAMPLE B-9

Powdered Soy Sauce

While 3.5 parts by weight of an anhydrous crystallinecyclotetrasaccharide, obtained by the method in Example A-3, and 0.02part by weight of crystalline cyclotetrasaccharide, penta- orhexa-hydrate, obtained by the method in Experiment 20, were freely-movedover a conveyer, one part by weight of “koikuchi-shoyu” (a pale-coloredsoy sauce) was sprayed over the mixture. The resulting mixture wastransferred to an aging tower and allowed to stand at 30° C. overnightto obtain a powdered soy sauce while converting the saccharide intocrystalline cyclotetrasaccharide, penta- or hexa-hydrate. The productcan be arbitrarily used as a seasoning for instant soups.

EXAMPLE B-10

Powdered Egg Yolk

Yolks prepared from fresh eggs were sterilized by a plate heatingsterilizer at 60 to 64° C., and one part by weight of the resultingliquid of egg yolks was mixed with 3.5 parts by weight of an anhydrouscrystalline cyclotetrasaccharide, obtained by the method in Example A-1,and similarly as in Example B-7, the mixture was shaped into a block,followed by pulverizing the block into a powdered egg yolk. The productcan be used as a material for confectioneries such as premixes, frozendeserts, and emulsifiers, as well as diets of weaning and therapeuticnutrients such as oral fluid diets and foods for intubation feedings.Also it can be used as a skin-beautifying agent or hair restorer.

EXAMPLE B-11

Powdered Yogurt

One part by weight of a plain yogurt was mixed with 3.6 parts by weightof an anhydrous amorphous cyclotetrasaccharide, obtained by the methodin Example A-3 and, similarly as in Example B-7, the mixture was shapedinto a block, followed by pulverizing the block into a powdered yogurt.The product can be used as a material for confectioneries such aspremixes, frozen deserts, and emulsifiers, as well as diets of weaningand therapeutic nutrients such as oral fluid diets and foods forintubation feedings. Also it can be arbitrarily incorporated intomargarines, whipping creams, spreads, cheese cakes, and jellies intoyogurt-flavored products. The powdered yogurt in this example can beshaped by a granulator or a tabletting machine into a product withlactic acid bacteria for use as an intestinal controlling agent.

EXAMPLE B-12

Hot Cake Mix

To 200 parts by weight of wheat flour were added 60 parts by weight of apowdered egg yolk obtained by the method in Example B-10, 25 parts byweight of butter, 10 parts by weight of sugar, 12 parts by weight of abaking powder, and 0.5 part by weight of salt to obtain a hot cake mix.After dissolving in water or milk, the product can be baked to easilyobtain a hot cake with a satisfactory taste and flavor.

EXAMPLE B-13

Powdered Ginseng Extract

A half part by weight of a ginseng extract was mixed with 1.2 parts byweight of an anhydrous crystalline cyclotetrasaccharide obtained by themethod in Example A-1 and, similarly as in Example B-7, the mixture wasshaped into a block and pulverized into a powdered ginseng extract. Theproduct was subjected to a granulator together with adequate amounts ofpowders of vitamins B₁ and B₂ to obtain a vitamin-containing granule ofginseng extract. The product thus obtained can be advantageously used asan agent for recovering healthy conditions from fatigue and for tonic,pickup, or hair restorer.

EXAMPLE B-14

Powdered Propolis Extract

A material propolis was extracted with a 95% (v/v) aqueous ethanolsolution in a usual manner, and the remaining residue was washed with asmall amount of water. The resulting extract and the water used forwashing the residues were pooled into an 80% (v/v) aqueous ethanolsolution as a crude propolis extract with a solid content of about 20%(w/w), d.s.b., which was then diluted with water to lower the ethanolconcentration to 50% (v/v). The resulting solution was kept at 50° C.for one hour to form an upper layer containing the effective ingredientsof propolis and a lower layer containing viscus sediments, and allowedto stand at ambient temperature overnight, followed by separating andcollecting the upper layer, i.e., a liquid propolis extract with asatisfactory color tint, flavor, and antimicrobial action in a yield ofabout 48%, d.s.b., to the crude propolis extract. One part by weight ofthe purified propolis extract was sprayed and mixed with 10 parts byweight of an anhydrous crystalline cyclotetrasaccharide obtained by themethod in Example A-1, and the resulting mixture was dried into apowdered propolis extract with a satisfactory flavor and taste. Theproduct can be used intact as an antiseptic, antioxidant,anti-inflammatory, immunoregulatory agent, or macrophage activatingagent; and mixed with other appropriate materials for use in foodproducts, cosmetics, and anti-susceptive diseases which can be treatedand/or prevented with the propolis extract.

EXAMPLE B-15

Powdered Extract of Japanese Indigo Plant

Thirty kilograms of terrestrial parts of an indigo plant, an annualplant of the genus Polygonum, having a botanical name of Polygonumtinctorium, were crushed, extracted with a 90% (v/v) aqueous ethanolsolution in a usual manner. The remaining residues were washed with asmall amount of water. The resulting extract and the water used forwashing the residues were pooled into an aqueous solution as a crudeindigo extract which was then diluted with water to lower the ethanolconcentration to 50% (v/v). One part by weight of the crude indigoextract was mixed with 12 parts by weight of crystallinecyclotetrasaccharide, monohydrate, obtained by the method in ExampleA-2. The mixture was transferred to a vat, allowed to stand for two daysto form a block while converting the saccharide into crystallinecyclotetrasaccharide, penta- or hexa-hydrate. The resulting block waspulverized by a cutter and classified to obtain a powdered indigoextract. The product has diversified physiological actions such as anantiseptic-, antiviral-, antitumor-, radical entrapping-, apoptosiscontrolling-, and cytokine regulatory-actions, and can be arbitrarilyused as a crude drug to be incorporated into food products, cosmetics,and pharmaceuticals.

EXAMPLE B-16

Powdered Coriander

Terrestrial parts of a coriander (Coriandrum sativum L.), a plant of thefamily Umbelliferae and the genus Coriandrum, were washed with water,drained, and cut into small pieces by a blender. The pieces were passedthrough a 150-mesh sieve using a centrifugal filtration separator,followed by collecting the extract and treated at 121° C. for 10 min toobtain a coriander extract containing 60 mg/ml solids. One part byweight of the extract was mixed with nine parts by weight of anhydrouscrystalline cyclotetrasaccharide in Example A-4, and the mixture wastransferred to a vat, allowed to stand for two days to form a blockwhile converting the saccharide into crystalline cyclotetrasaccharide,penta- or hexa-hydrate. The resulting block was pulverized by a cutterand classified to obtain a powdered coriander extract. The product hasan activity of inhibiting the adhesion of metals such as lead and can beadvantageously used intact or in combination with food products orpharmaceuticals.

EXAMPLE B-17

Powdered Royal Jelly

One part by weight of an intact Brazilian royal jelly with a moisturecontent of 65% (w/w) was mixed with seven parts by weight of anhydrouscrystalline cyclotetrasaccharide obtained by the method in Example A-1.The resulting mixture was transferred to a vat and allowed to stand fortwo days for forming a block while converting the saccharide intocrystalline cyclotetrasaccharide, penta- or hexa-hydrate. The block waspulverized by a cutter into a powdered royal jelly which was thenclassified by passing through a 100-mesh sieve and tabletted by atabletting machine to obtain tablets, 300 mg each. The product has astrong tonic action and cell activating action and stably retains theroyal jelly susceptible to deterioration for a relatively long period oftime even at ambient temperature. Since the product has an improvedflavor and taste, as well as a mild sweetness and an adequate sourtaste, it can be arbitrarily used as a health food for daily use.

EXAMPLE B-18

Solid Preparation for Fluid Food

A composition, consisting of 400 parts by weight of an anhydrouscrystalline cyclotetrasaccharide powder obtained by the method inExample A-1, 270 parts by weight of a powdered egg yolk obtained by themethod in Example B-7, 209 parts by weight of a skim milk powder, 4.4parts by weight of sodium chloride, 1.85 parts by weight of potassiumchloride, 0.01 part by weight of thiamine, 0.1 part by weight of sodiumL-ascorbate, 0.6 part by weight of vitamin E acetate, and 0.04 part byweight of nicotinic acid amid, was prepared. Twenty-five grams aliquotsof the composition were injected into moisture-proof laminated smallbags which were then heat sealed to obtain a solid preparation for fluidfoods.

The product, wherein the moisture content in its inner atmosphere islowed, has a relatively long shelf-live without a need of cold storage.Also it has a satisfactory dispersibility and solubility in water. Inuse, one bag of the product is dissolved in about 150–300 ml water intoa fluid food and then orally administered to a subject or intubationallyadministered to the nasal cavity, stomach, intestine, etc.

EXAMPLE B-19

Tablet Preparation for Medical Use

New born hamsters were injected with an antiserum prepared from rabbitsby a conventional method to reduce their immunoreaction, subcutaneouslytransplanted with BALL-1 cells, and bred for three weeks in a usualmanner. Tumor masses formed subcutaneously were extracted, cut intopieces, and suspended in physiological saline. The resulting cellsuspension was washed with RPMI 1640 medium (pH 7.2) free of serum,suspended in a fresh preparation of the same medium to give a celldensity of about 2×10⁶ cells/ml, and incubated at 35° C.

After the addition of 200 IU/ml of a partially purified humaninterferon-α, the cell suspension was incubated for about two hours andthen admixed with about 300 HA/ml of sendai virus (HVJ) and incubatedfor 20 hours to induce human interferon-α. The resulting culture wascentrifuged at 4° C. and about 1,000×g, followed by removing sediments.The resulting supernatant was membrane filtered, and the filtrate was ina conventional manner fed to a column immobilized with ananti-interferon-α antibody, followed by removing non-adsorbed fractions.The interferon adsorbed on the antibody was eluted as adsorbed fractionswhich were then concentrated with a membrane into a 4-ml concentrate,containing about 0.001% (w/v) proteins and a human interferon-α with aspecific activity of about 2×10⁸ IU/mg protein, per hamster.

One kilogram of anhydrous crystalline cyclotetrasaccharide, obtained bythe method in Example A-1, was pulverized, passed through a 150-meshsieve, and mixed to homogeneity with a dilute, which had been preparedby diluting 0.25 ml of the above concentrate having about 1×10⁶ IU ofinterferon-α with 100 ml distilled water, while spraying the dilute overthe saccharide powder. The resulting mixture was in a usual mannertabletted by a tabletting machine to obtain a 300 mg tablet with 150 IUof interferon-α. The process in this example easily dehydrates solutionsof interferon-α only by spraying an anhydrous crystallinecyclotetrasaccharide powder, facilitates homogeneous mixing, and alsoeffectively stabilizes the interferon-α.

Since the product easily dissolves in water, it can be advantageouslyused as an agent for anti-susceptive diseases, which can be treatedand/or prevented with interferon-α, such as an antiviral-, antitumor-,antirheumatic-, and anti-immunopathic-agents in the form of an internalor oral agent. Also the product can be advantageously used as a reagentfor examination.

EXAMPLE B-20

Granular Preparation for Medical Use

A stock culture of BALL-1 cell, a human lymphoblastoid cell line, wasinoculated into Eagle's minimum essential medium (pH 7.4) supplementedwith 20% (v/v), and in a usual manner subjected to an in vitrosuspension culture at 37° C. The resulting cells were washed withEagle's minimum essential medium (pH 7.4) free of serum and suspended ina fresh preparation of the same medium to give a cell density of about1×10⁷ cells/ml. HVJ was added to the cell suspension in an amount ofabout 1,000 HA/ml and incubated at 38° C. for one day to induce tumornecrosis factor-α (TNF-α). The resulting culture was centrifuged at 4°C. and about 1,000×g, and the supernatant was dialyzed for 15 hoursagainst 0.01 M phosphate buffer (pH 7.2) in physiological saline, andmembrane filtered. The filtrate was in a usual manner fed to a column ofanti-interferon antibody, and the non-adsorbed fractions were subjectedto affinity chromatography using a column packed with gels of anti-TNF-αmonoclonal antibody to purify the formed TNF-α, followed byconcentrating the desired fractions to obtain a concentrate having aprotein concentration of about 0.01% (w/v) and TNF-α with a specificactivity of about 2×10⁶ JRU/mg protein in a yield of about 5×10⁴ JRU perL of the culture after the induction and formation of TNF-α.

A half milliliter of the above TNF-α concentrate with TNF-α activity ofabout 1×10⁵ JRU was diluted with 100 ml of distilled water and thenmixed to homogeneity with a cyclotetrasaccharide powder, which had beenprepared by pulverizing one kilogram of anhydrous crystallinecyclotetrasaccharide obtained by the method in Example A-1, and passingthrough a 150-mesh sieve, while spraying the dilute over the powder. Theresulting mixture was then in a usual manner granulated by a granulatorinto a TNF-α preparation in the form of a granule, containing about 100JRU/g of TNF-α. The process in this example easily dehydrates solutionsof TNF-α only by spraying an anhydrous crystalline cyclotetrasaccharidepowder, facilitates homogeneous mixing, and also effectively stabilizesthe TNF-α.

Since the product easily dissolves in water, it can be advantageouslyused as an agent for anti-susceptive diseases, which can be preventedand/or treated with TNF-α, such as an antiviral-, antitumor-,antirheumatic-, and anti-immunopathic-agents in the form of an internalor oral agent. Also the product can be advantageously used as a reagentfor examination.

EXAMPLE B-21

Ointment for Traumatherapy

Four hundred parts by weight of anhydrous crystallinecyclotetrasaccharide obtained by the method in Example A-1 were admixedwith three parts by weigh of iodine dissolved previously in 50 parts byweight of methanol, and further admixed with 200 parts by weight of a10% aqueous pullulan solution and 50 parts by weight of crystallinemaltose hydrate. The resulting mixture was allowed to stand at ambienttemperature overnight to convert the cyclotetrasaccharide intocrystalline cyclotetrasaccharide, penta- or hexa-hydrate, to obtain anointment for traumatherapy with an adequate adhesiveness andextendibility.

By applying to affected skin parts directly or after pasted on gauzes,oilpapers or the like, the product cures external injuries such as skinulcers induced by cuts, excoriations, burns, and dermatophytosises(athlete's foot).

POSSIBILITY OF INDUSTRIAL APPLICABILITY

As evident from the above, the present invention relates to adehydrating agent comprising a non-reducing cyclotetrasaccharide as aneffective ingredient, particularly, cyclotetrasaccharide withdehydrating ability can be advantageously used, as the effectiveingredient, to reduce the moisture content in the inner atmosphere ofmoisture-proof containers which house dried food products, etc., and inhydrous products such as food products, cosmetics, pharmaceuticals,industrial chemicals, and their materials and processing intermediates.The method of the present invention, which comprises a step of allowinga cyclotetrasaccharide with dehydrating ability to contact with hydrousmatters to substantially reduce their moisture content through theconversion of the cyclotetrasaccharide into crystallinecyclotetrasaccharide, penta- or hexa-hydrate. Since the method does notneed severe conditions such as heat drying, it can easily dehydrate thefollowing hydrous matters and facilitates to produce high qualitydehydrated products: Examples of the above hydrous matters include foodproducts susceptible to deteriorating flavor and taste, andpharmaceuticals with effective ingredients susceptible to decomposingand lowering their activities. The dehydrated products, which are wellprevented from bacterial contamination and which the denaturalization ordeterioration such as hydrolysis, rancidity, and browning are inhibited,have a relatively long, stable shelf-life.

The present invention with such outstanding effects and functions is asignificant invention that greatly contributes to this art.

1. A method for producing a dehydrated product, which comprises a stepof either incorporating, contacting or coexisting a saccharide havingthe structure of cyclo{6)-α-D-glucopyranosyl-(1 3)-α-D-glucopyransyl-(16)-α-D-glucopyranosyl-(1 3)-α-D-glucopyranosyl-(1}, into, with, or in afood product, cosmetic, pharmaceutical, or industrial chemicalcontaining water, wherein said saccharide is in a crystalline anhydrousform, crystalline monohydrous form or anhydrous amorphous form.
 2. Themethod of claim 1, where the saccharide is in an amount of in the rangeof 0.001–200 parts by weight to one part by weight of the food product,cosmetic, pharmaceutical, or industrial chemical containing water.